Anti-cancer fusion polypeptide

ABSTRACT

The disclosure provides a fusion polypeptide specific for both CD137 and HER2/neu, which fusion polypeptide can be useful for directing CD137 clustering and activation to HER2/neu-positive tumor cells. Such fusion polypeptide can be used in many pharmaceutical applications, for example, as anti-cancer agents and/or immune modulators for the treatment or prevention of human diseases such as a variety of tumors. The present disclosure also concerns methods of making the fusion polypeptide described herein as well as compositions comprising such fusion polypeptide. The present disclosure further relates to nucleic acid molecules encoding such fusion polypeptide and to methods for generation of such fusion polypeptide and nucleic acid molecules. In addition, the application discloses therapeutic and/or diagnostic uses of such fusion polypeptide as well as compositions comprising one or more of such fusion polypeptides.

I. BACKGROUND

HER2/neu is a member of the human epidermal growth factor receptorfamily. Amplification or overexpression of this oncogene has been shownto play an important role in the development and progression of avariety of tumors, including certain aggressive types of breast cancer.HER2/neu has been shown to be highly differentially expressed on tumorcells with much higher cell-surface density compared to healthy tissue.

Trastuzumab (marketed as Herceptin®), a monoclonal antibody targetingHER2/neu, is indicated for the treatment of women with either earlystage or metastatic HER2(+) breast cancer. Despite the promisingactivity of monoclonal antibodies such as Trastuzumab in this setting,the response rates among patients with either refractory or advancedcancer are suboptimal. For example, while the objective response raterose significantly in a clinical trial comparing chemotherapy alone withchemotherapy plus Trastuzumab—from 32% to 50% —, this still left theother half of the enrolled patients having no response (Slamon D. J. etal., N Engl J Med. 2001 Mar. 15; 344(11):783-92). Therefore, betterHER2/neu-targeting therapies with an improved response rate arerequired.

CD137 is a co-stimulatory immune receptor and a member of the tumornecrosis factor receptor (TNFR) super-family. It is mainly expressed onactivated CD4+ and CD8+ T cells, activated B cells, and natural killer(NK) cells but can also be found on resting monocytes and dendriticcells (Li, S. Y. et al., Clin Pharmacol 2013 5(Suppl 1):47-53), orendothelial cells (Snell, L. M. et al., Immunol Rev 2011 November;244(1):197-217). CD137 plays an important role in the regulation ofimmune responses and thus is a target for cancer immunotherapy. CD137ligand (CD137L) is the only known natural ligand of CD137, and isconstitutively expressed on several types of APC, such as activated Bcells, monocytes, and splenic dendritic cells, and it can be induced onT lymphocytes.

CD137L is a trimeric protein that exists as a membrane-bound form and asa soluble variant. The ability of soluble CD137L to activate CD137 e.g.on CD137-expressing lymphocytes is limited, however, and largeconcentrations are required to elicit an effect (Wyzgol, A. et al., JImmunol 2009 Aug. 1; 183(3):1851-1861). The natural way of activation ofCD137 is via the engagement of a CD137-positive cell with aCD137L-positive cell. CD137 activation is then thought to be induced byclustering through CD137L on the opposing cell, leading to signaling viaTRAF1, 2 and 3 (Snell, L. M. et al., Immunol Rev 2011 November;244(1):197-217, Yao, S. et al., Nat Rev Drug Disc 2013 February;12(2):130-146) and further concomitant downstream effects in theCD137-positive T-cell. In the case of T-cells activated by recognitionof their respective cognate targets, the effects elicited bycostimulation of CD137 are a further enhanced activation, enhancedsurvival and proliferation, the production of pro-inflammatory cytokinesand an improved capacity to kill.

The benefit of CD137 costimulation for the elimination of cancer cellshas been demonstrated in a number of preclinical in-vivo models. Theforced expression of CD137L on a tumor, for example, leads to tumorrejection (Melero, I. et al., Eur J Immunol 1998 March;28(3):1116-1121). Likewise, the forced expression of an anti-CD137 scFvon a tumor leads to a CD4⁺ T-cell and NK-cell dependent elimination ofthe tumor (Ye, Z. et al., Nat Med 2002 April; 8(4):343-348, Zhang, H. etal., Mol Canc Ther 2006 January; 5(1):149-155, Yang, Y. et al., Canc Res2007 Mar. 1; 67(5):2339-2344). A systemically administered anti-CD137antibody has also been demonstrated to lead to retardation of tumorgrowth (Martinet, O. et al., Gene Ther 2002 June; 9(12):786-792).

It has been shown that CD137 is an excellent marker for naturallyoccurring tumor-reactive T cells in human tumors (Ye, Q. et al., ClinCanc Res: 2014 Jan. 1; 20(1):44-55), and that anti-CD137 antibodies canbe employed to improve the expansion and activity of CD8+ melanomatumor-infiltrating lymphocytes for the application in adoptive T-celltherapy (Chacon, J. A. et al., PloS One 2013 8(4):e60031).

The preclinical demonstration of the potential therapeutic benefit ofCD137 costimulation has spurred the development of therapeuticantibodies targeting CD137, BMS-663513 (Jure-Kunkel, M. et al., U.S.Pat. No. 7,288,638) and PF-05082566 (Fisher, T. S. et al., Canc ImmunolImmunother 2012 October; 61(10):1721-1733); both are currently in earlyclinical trials.

However, it has only recently been appreciated that a bivalentCD137-binder like an antibody may by itself not be sufficient to clusterCD137 on T-cells or NK-cells and lead to efficient activation, inanalogy to the lack of activity of the trivalent soluble CD137L. Inrecent publications utilizing preclinical mouse models, in-vivo evidencehas been presented that the mode of action of other anti-TNFR antibodiesin fact requires the interaction of the antibodies via their Fc-partwith Fc-gamma receptors on Fc-gamma-receptor expressing cells (Bulliard,Y. et al., J Exp Med 2013 Aug. 26; 210(9):1685-1693, Bulliard, Y. etal., Immunol Cell Biol 2014 July; 92(6):475-480). The mode of action ofthe antibodies currently in clinical development may therefore bedominated by a non-targeted clustering via Fc-gamma receptors which maybe nearly randomly dependent on the presence of Fc-γ-expressing cells inthe vicinity of the tumor.

Thus, there is unmet need for the generation of therapeutics thatcluster and activate CD137 with a specific tumor-targeted mode ofaction.

To meet this unmet need, the present application, provides a novelapproach of simultaneously engaging CD137 and tumor antigen HER2/neu viaa fusion polypeptide having the following properties:

(a) binding specificity for CD137; and(b) binding specificity for HER2/neu;

This fusion polypeptide is designed to provide a tumor-target-dependentactivation of CD137 on lymphocytes, via HER2 overexpressed on tumorcells. Such a molecule is expected to further activate T-cells and/or NKcells that are located in the vicinity of a HER2-positive tumor. Such abispecific may display improved therapeutic effects over eitheranti-HER2 or anti-CD137 antibodies.

II. Definitions

The following list defines terms, phrases, and abbreviations usedthroughout the instant specification. All terms listed and definedherein are intended to encompass all grammatical forms.

As used herein, unless otherwise specified, “CD137” means human CD137.CD137 is also known as “4-1BB” or “tumor necrosis factor receptorsuperfamily member 9 (TNFRSF9)” or “induced by lymphocyte activation(ILA)”. Human CD137 means a full-length protein defined by UniProtQ07011, a fragment thereof, or a variant thereof.

As used herein, unless otherwise specified, “HER2” or “HER2/neu” meanshuman HER2. Her-2 or HER2/neu is also known as “erbB-2”, “c-neu”, or“p185”. Human Her 2 means a full-length protein defined by UniProtP04626, a fragment thereof, or a variant thereof.

As used herein, “detectable affinity” means the ability to bind to aselected target with an affinity constant of generally at least about10⁻⁵ M or below. Lower affinities are generally no longer measurablewith common methods such as ELISA and therefore of secondary importance.

As used herein, “binding affinity” of a protein of the disclosure (e.g.a mutein of a lipocalin) or a fusion polypeptide thereof to a selectedtarget (in the present case, CD137 and/or HER2/neu), can be measured(and thereby KD values of a mutein-ligand complex be determined) by amultitude of methods known to those skilled in the art. Such methodsinclude, but are not limited to, fluorescence titration, competitionELISA, calorimetric methods, such as isothermal titration calorimetry(ITC), and surface plasmon resonance (BIAcore). Such methods are wellestablished in the art and examples thereof are also detailed below.

It is also noted that the complex formation between the respectivebinder and its ligand is influenced by many different factors such asthe concentrations of the respective binding partners, the presence ofcompetitors, pH and the ionic strength of the buffer system used, andthe experimental method used for determination of the dissociationconstant K_(D) (for example fluorescence titration, competition ELISA orsurface plasmon resonance, just to name a few) or even the mathematicalalgorithm which is used for evaluation of the experimental data.

Therefore, it is also clear to the skilled person that the K_(D) values(dissociation constant of the complex formed between the respectivebinder and its target/ligand) may vary within a certain experimentalrange, depending on the method and experimental setup that is used fordetermining the affinity of a particular lipocalin mutein for a givenligand. This means that there may be a slight deviation in the measuredK_(D) values or a tolerance range depending, for example, on whether theK_(D) value was determined by surface plasmon resonance (Biacore), bycompetition ELISA, or by “direct ELISA.”

As used herein, a “mutein,” a “mutated” entity (whether protein ornucleic acid), or “mutant” refers to the exchange, deletion, orinsertion of one or more nucleotides or amino acids, compared to thenaturally occurring (wild-type) nucleic acid or protein “reference”scaffold. Said term also includes fragments of a mutein and variants asdescribed herein. Lipocalin muteins of the present invention, fragmentsor variants thereof preferably retain the function of binding to CD137as described herein.

The term “fragment” as used herein in connection with the muteins of thedisclosure relates to proteins or peptides derived from full-lengthmature human tear lipocalin that are N-terminally and/or C-terminallyshortened, i.e. lacking at least one of the N-terminal and/or C-terminalamino acids. Such fragments may include at least 10, more such as 20 or30 or more consecutive amino acids of the primary sequence of the maturelipocalin and are usually detectable in an immunoassay of the maturelipocalin. In general, the term “fragment”, as used herein with respectto the corresponding protein ligand CD137 of a lipocalin mutein of thedisclosure or of the combination according to the disclosure or of afusion protein described herein, relates to N-terminally and/orC-terminally shortened protein or peptide ligands, which retain thecapability of the full length ligand to be recognized and/or bound by amutein according to the disclosure.

The term “mutagenesis” as used herein means that the experimentalconditions are chosen such that the amino acid naturally occurring at agiven sequence position of the mature lipocalin can be substituted by atleast one amino acid that is not present at this specific position inthe respective natural polypeptide sequence. The term “mutagenesis” alsoincludes the (additional) modification of the length of sequencesegments by deletion or insertion of one or more amino acids. Thus, itis within the scope of the disclosure that, for example, one amino acidat a chosen sequence position is replaced by a stretch of three randommutations, leading to an insertion of two amino acid residues comparedto the length of the respective segment of the wild-type protein. Suchan insertion or deletion may be introduced independently from each otherin any of the peptide segments that can be subjected to mutagenesis inthe disclosure. In one exemplary embodiment of the disclosure, aninsertion of several mutations may be introduced into the loop AB of thechosen lipocalin scaffold (cf. International Patent Application WO2005/019256 which is incorporated by reference its entirety herein).

The term “random mutagenesis” means that no predetermined single aminoacid (mutation) is present at a certain sequence position but that atleast two amino acids can be incorporated with a certain probability ata predefined sequence position during mutagenesis.

“Identity” is a property of sequences that measures their similarity orrelationship. The term “sequence identity” or “identity” as used in thepresent disclosure means the percentage of pair-wise identicalresidues—following (homologous) alignment of a sequence of a polypeptideof the disclosure with a sequence in question—with respect to the numberof residues in the longer of these two sequences. Sequence identity ismeasured by dividing the number of identical amino acid residues by thetotal number of residues and multiplying the product by 100.

The term “homology” is used herein in its usual meaning and includesidentical amino acids as well as amino acids which are regarded to beconservative substitutions (for example, exchange of a glutamate residueby an aspartate residue) at equivalent positions in the linear aminoacid sequence of a polypeptide of the disclosure (e.g., any lipocalinmutein of the disclosure).

The percentage of sequence homology or sequence identity can, forexample, be determined herein using the program BLASTP, version blastp2.2.5 (Nov. 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res.25, 3389-3402). In this embodiment the percentage of homology is basedon the alignment of the entire polypeptide sequences (matrix: BLOSUM 62;gap costs: 11.1; cutoff value set to 10-) including the propeptidesequences, preferably using the wild-type protein scaffold as referencein a pairwise comparison. It is calculated as the percentage of numbersof “positives” (homologous amino acids) indicated as result in theBLASTP program output divided by the total number of amino acidsselected by the program for the alignment.

Specifically, in order to determine whether an amino acid residue of theamino acid sequence of a lipocalin (mutein) different from a wild-typelipocalin corresponds to a certain position in the amino acid sequenceof a wild-type lipocalin, a skilled artisan can use means and methodswell-known in the art, e.g., alignments, either manually or by usingcomputer programs such as BLAST2.0, which stands for Basic LocalAlignment Search Tool or ClustalW or any other suitable program which issuitable to generate sequence alignments. Accordingly, a wild-typelipocalin can serve as “subject sequence” or “reference sequence”, whilethe amino acid sequence of a lipocalin different from the wild-typelipocalin described herein serves as “query sequence”. The terms“reference sequence” and “wild-type sequence” are used interchangeablyherein. A preferred wild-type lipocalin is shown in SEQ ID NO: 18 (Tlc)or SEQ ID NO: 17 (NGAL), respectively. Dependent on whether a lipocalinmutein of the present invention is based on Tlc or NGAL, respectively,the corresponding wild-type lipocalin may be used as reference sequenceor wild-type sequence.

“Gaps” are spaces in an alignment that are the result of additions ordeletions of amino acids. Thus, two copies of exactly the same sequencehave 100% identity, but sequences that are less highly conserved, andhave deletions, additions, or replacements, may have a lower degree ofsequence identity. Those skilled in the art will recognize that severalcomputer programs are available for determining sequence identity usingstandard parameters, for example Blast (Altschul, et al. (1997) NucleicAcids Res. 25, 3389-3402), Blast2 (Altschul, et al. (1990) J. Mol. Biol.215, 403-410), and Smith-Waterman (Smith, et al. (1981) J. Mol. Biol.147, 195-197).

The term “variant” as used in the present disclosure relates toderivatives of a protein or peptide that include modifications of theamino acid sequence, for example by substitution, deletion, insertion orchemical modification. Such modifications do in some embodiments notreduce the functionality of the protein or peptide. Such variantsinclude proteins, wherein one or more amino acids have been replaced bytheir respective D-stereoisomers or by amino acids other than thenaturally occurring 20 amino acids, such as, for example, ornithine,hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline.However, such substitutions may also be conservative, i.e. an amino acidresidue is replaced with a chemically similar amino acid residue.Examples of conservative substitutions are the replacements among themembers of the following groups: 1) alanine, serine, and threonine; 2)aspartic acid and glutamic acid; 3) asparagine and glutamine; 4)arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and6) phenylalanine, tyrosine, and tryptophan. The term “variant”, as usedherein with respect to the corresponding protein ligand CD137 of alipocalin mutein of the disclosure or of the combination according tothe disclosure or of a fusion protein described herein, relates to CD137or fragment thereof, respectively, that has one or more such as 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50,60, 70, 80 or more amino acid substitutions, deletions and/or insertionsin comparison to a wild-type CD137 protein, respectively, such as aCD137 reference protein as deposited with UniProt as described herein. ACD137 variant, respectively, has preferably an amino acid identity of atleast 50%, 60%, 70%, 80%, 85%, 90% or 95% with a wild-type human CD137,such as a CD137 reference protein as deposited with UniProt as describedherein.

By a “native sequence” lipocalin is meant a lipocalin that has the sameamino acid sequence as the corresponding polypeptide derived fromnature. Thus, a native sequence lipocalin can have the amino acidsequence of the respective naturally-occurring lipocalin from anyorganism, in particular a mammal. Such native sequence polypeptide canbe isolated from nature or can be produced by recombinant or syntheticmeans. The term “native sequence” polypeptide specifically encompassesnaturally-occurring truncated or secreted forms of the lipocalin,naturally-occurring variant forms such as alternatively spliced formsand naturally-occurring allelic variants of the lipocalin. A polypeptide“variant” means a biologically active polypeptide having at least about50%, 60%, 70%, 80% or at least about 85% amino acid sequence identitywith the native sequence polypeptide. Such variants include, forinstance, polypeptides in which one or more amino acid residues areadded or deleted at the N- or C-terminus of the polypeptide. Generally avariant has at least about 70%, including at least about 80%, such as atleast about 85% amino acid sequence identity, including at least about90% amino acid sequence identity or at least about 95% amino acidsequence identity with the native sequence polypeptide. As anillustrative example, the first 4 N-terminal amino acid residues(His-His-Leu-Leu) and the last 2 C-terminal amino acid residues(Ser-Asp) can be deleted in a tear lipocalin (Tlc) mutein of thedisclosure without affecting the biological function of the protein,e.g. SEQ ID NOs: 32-38. In addition, as another illustrative example,certain amino acid residues can be deleted in a lipocalin 2 (NGAL)mutein of the disclosure without affecting the biological function ofthe protein, e.g. (Lys-Asp-Pro, positions 46-48) as to SEQ ID NO: 42.

The term “position” when used in accordance with the disclosure meansthe position of either an amino acid within an amino acid sequencedepicted herein or the position of a nucleotide within a nucleic acidsequence depicted herein. To understand the term “correspond” or“corresponding” as used herein in the context of the amino acid sueqnecepositions of one or more lipocalin muteins, a corresponding position isnot only determined by the number of the preceding nucleotides/aminoacids. Accordingly, the position of a given amino acid in accordancewith the disclosure which may be substituted may vary due to deletion oraddition of amino acids elsewhere in a (mutant or wild-type) lipocalin.Similarly, the position of a given nucleotide in accordance with thepresent disclosure which may be substituted may vary due to deletions oradditional nucleotides elsewhere in a mutein or wild-type lipocalin5-untranslated region (UTR) including the promoter and/or any otherregulatory sequences or gene (including exons and introns).

Thus, for a corresponding position in accordance with the disclosure, itis preferably to be understood that the positions of nucleotides/aminoacids may differ in the indicated number than similar neighbouringnucleotides/amino acids, but said neighbouring nucleotides/amino acids,which may be exchanged, deleted, or added, are also comprised by the oneor more corresponding positions.

In addition, for a corresponding position in a lipocalin mutein based ona reference scaffold in accordance with the disclosure, it is preferablyto be understood that the positions of nucleotides/amino acids arestructurally corresponding to the positions elsewhere in a (mutant orwild-type) lipocalin, even if they may differ in the indicated number,as appreciated by the skilled in light of the highly-conserved overallfolding pattern among Iipocalins.

The word “detect”, “detection”, “detectable” or “detecting” as usedherein is understood both on a quantitative and a qualitative level, aswell as a combination thereof. It thus includes quantitative,semi-quantitative and qualitative measurements of a molecule ofinterest.

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman. The term “mammal” is used herein to refer to any animalclassified as a mammal, including, without limitation, humans, domesticand farm animals, and zoo, sports, or pet animals, such as sheep, dogs,horses, cats, cows, rats, pigs, apes such as cynomolgous monkeys andetc., to name only a few illustrative examples. Preferably, the mammalherein is human.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations.

A “sample” is defined as a biological sample taken from any subject.Biological samples include, but are not limited to, blood, serum, urine,feces, semen, or tissue.

A “subunit” of a fusion polypeptide disclosed herein is defined as astretch of amino acids of the polypeptide, which stretch defines aunique functional unit of said polypeptide such as provides bindingmotif towards a target.

III. DESCRIPTIONS OF FIGURES

FIG. 1: provides an overview over the design of the representativefusion polypeptides described in this application, which are bispecificwith regard to the targets, HER2 and CD137. Representative fusionpolypeptides were made based on an antibody specific for HER2 (SEQ IDNOs: 3 and 4) and a lipocalin mutein specific for CD137 (SEQ ID NO: 2).Direct fusion of the antibody with the lipocalin mutein resulted in afusion polypeptide of SEQ ID NOs: 5 and 6. Lipocalin muteins were fusedto either one of the four termini of the antibody, using an engineeredIgG4 backbone with the mutations S228P, F234A and L235A (SEQ ID NOs: 9and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and16). In addition, within the fusion polypeptide SEQ ID NOs: 7 and 8), aN297A mutation was made to remove the glycosylation motif.

FIG. 2: depicts the results of an ELISA experiment in which the affinityof representative fusion polypeptides and the benchmark antibody againstHER2 was determined. Recombinant HER2 was coated on a microtiter plate,and the tested agents were titrated starting from a concentration of 100nM. Bound agents under study were detected via an anti-human IgG Fcantibody as described in Example 2. The data was fit with a 1:1 bindingmodel with EC50 value and the maximum signal as free parameters, and aslope that was fixed to unity. The resulting EC50 values are provided inTable 2.

FIG. 3: shows the results of an ELISA experiment in which the affinityof representative fusion polypeptides and the positive control lipocalinmutein against CD137 was determined. An Fc-fusion of human CD137 wascoated on a microtiter plate, and the tested agents were titratedstarting from a concentration of 100 nM. Bound agents under study weredetected via an anti-human-IgG-Fc antibody as described in Example 3.The data was fit with a 1:1 binding model with EC50 value and themaximum signal as free parameters, and a slope that was fixed to unity.The resulting EC50 values are provided in Table 3.

FIG. 4: illustrates the results of an ELISA experiment in which theability of representative fusion polypeptides to simultaneously bindboth targets, HER2 and CD137, was determined. Recombinant HER2 wascoated on a microtiter plate, followed by a titration of the fusionpolypeptides starting from a concentration of 100 nM. Subsequently, aconstant concentration of biotinylated human CD137-Fc was added, whichwas detected via extravidin as described in Example 4.

FIG. 5: shows the result of a T-cell activation assay in which theability of the fusion polypeptide of SEQ ID NOs: 15 and 16 toco-stimulate T-cell responses was assessed. The fusion polypeptide ofSEQ ID NOs: 15 and 16 at different concentrations was coated onto aplastic dish together with an anti-human CD3 antibody, and purifiedT-cells were subsequently incubated on the coated surface. Supernatantinterleukin 2 (IL-2) levels were measured as described in Example 5.

FIG. 6: provides a representative experiment in which the ability of thefusion polypeptide of SEQ ID NOs: 9 and 10 to co-stimulate T-cellactivation in a HER2-target-dependent manner was investigated. As acontrol, we employed the monospecific, HER2-binding antibody of SEQ IDNOs: 3 and 4. In the experiment, an anti-human CD3 antibody was coatedon a plastic culture dish, and subsequently HER2-positive SKBR3 cellswere cultured on the dish overnight. The next day, purified T-cells wereincubated on the coated surface in the presence of variousconcentrations of the bispecific fusion polypeptide SEQ ID NOs: 9 and 10(filled circles) or the control antibody of SEQ ID NOs: 3 and 4. Thevalues determined for SEQ ID NOs: 3 and 4 at different concentrationsare provided as the average (dotted line). Supernatant interleukin 2(IL-2) (A) and IFN-γ (B) were determined by anElectrochemoluminescence-based assay. The experiment was also performedin the presence of an excess of SEQ ID NOs: 3 and 4, and supernatantlevels of IL-2 (C) and IFN-γ (D) were measured. The data was fitted witha 1:1 binding model.

FIG. 7: provides a representative experiment in which the ability of thefusion polypeptide of SEQ ID NOs: 11 and 12 to co-stimulate T-cellactivation in a HER2-target-dependent manner was investigated. Fordetails; see legend of FIG. 6.

FIG. 8: provides a representative experiment in which the ability of thefusion polypeptide of SEQ ID NOs: 13 and 14 to co-stimulate T-cellactivation in a HER2-target-dependent manner was investigated. Fordetails; see legend of FIG. 6.

FIG. 9: provides a representative experiment in which the ability of thefusion polypeptide of SEQ ID NOs: 15 and 16 to co-stimulate T-cellactivation in a HER2-target-dependent manner was investigated. Fordetails; see legend of FIG. 6.

FIG. 10: provides a representative experiment on the affinity ofpolypeptides to FcgRI, FcgRIII and FcRn as described in Examples 7 and8.

FIG. 11: provides a representative experiment in which the ability ofthe fusion polypeptides indicated in the Figure to co-stimulate T-cellactivation with different cell lines was investigated. Cell linesutilized were the highly HER2-positive cells (SKBR3, BT474) and celllines expressing HER2 at a level similar to that of healthy cells(HepG2, MCF7). In the experiment, an anti-human CD3 antibody was coatedon a plastic culture dish, and subsequently the cell line under studywas cultured on the dish overnight. The next day, purified T-cells wereincubated on the coated surface for three days in the presence ofvarious concentrations of the bispecific fusion polypeptides as follows:(A) SEQ ID NOs: 9 and 10 (solid lines) or the control antibody of SEQ IDNOs: 3 and 4 (broken line). (B) Anti-CD137 antibody SEQ ID NOs: 32 and33. (C) Anti-CD137 antibody SEQ ID NOs: 34 and 35. Supernatantinterleukin 2 levels were determined by anElectrochemoluminescence-based assay. The plotted relative IL-2 responsecorresponds to the ratio of the responses obtained in the presence andin the absence (“background”) of test articles.

FIG. 12: provides representative size exclusion chromatography (SEC)traces of bispecific fusion polypeptides and the control antibody of SEQID NOs: 3 and 4 before (bottom curves) and after (top curves) incubationfor 4 weeks at 40° C. in PBS, pH 7.4. Sample concentration was 20 mg/mLin each case, fusion polypeptide identity was as indicated in thefigure. SEC curves are plotted with an offset on the y-axis for bettervisualization.

FIG. 13: provides the result of a pharmacokinetic analysis of thebispecific fusion polypeptides and the control antibody of SEQ ID NOs: 3and 4 in mice. Male CD-1 mice (3 mice per timepoint) were injectedintravenously with fusion polypeptides at a dose of 10 mg/kg. Druglevels were detected using a Sandwich ELISA detecting the fullbispecific construct via the targets HER2 and CD137. Trastuzumab plasmalevels were determined using a Sandwich ELISA with targets HER2 andhuman Fc. The data were fitted using a two-compartmental model.

FIG. 14: provides the result of a pharmacokinetic analysis of thebispecific fusion polypeptides and the control antibody of SEQ ID NOs: 3and 4 in mice. Male, trastuzumab-naïve cynomolgus monkeys received testarticles as an intravenous infusion of 60 minutes duration at a dose of3 mg/kg. Drug levels were detected using a Sandwich ELISA detecting thefull bispecific construct via the targets HER2 and CD137. Trastuzumabplasma levels were determined using a Sandwich ELISA with targets HER2and human Fc. The data were fitted using a two-compartmental model.

FIG. 15: provides the result of an in vitro T cell immunogenicityassessment of the bispecific fusion polypeptides, the control antibodyof SEQ ID NOs: 3 and 4 and the positive control keyhole limpethemocyanine (KLH). The assay was performed using a PBMC-based format asdescribed in Example 13, with 32 donors and human leukocyte antigen(HLA) allotypes reflective of the distribution in a global population:(A) Stimulation index (proliferation in the presence vs. absence of testarticle). The average responses are indicated as bars. The thresholdthat defines a responding donor (stimulation index >2) is indicated as adotted line. (B) Number of responders

FIG. 16: Relative median tumor volume after treatment with CD137/HER2bispecifics or controls in humanized mouse tumor model. NSG mice wereengrafted with s.c. SK-OV-3 tumors which were allowed to grow to anaverage of 120 mm3. Mice were randomized into treatment groups andreceived 7×106 fresh human PBMC i.v. and the molecules and dosesindicated 1 hour after PBMC injection on day 0, and again on day 7 andday 14. Each group contained 10 mice with the exception of the groupstudying SEQ ID NOs: 32 and 33 which consisted of 7 mice. Tumor growthwas recorded every 3-4 days.

FIG. 17: provides a representative experiment in which the ability ofthe fusion polypeptides indicated in the Figure to activate the CD137pathway in dependence of the HER2^(high) NCI-N87 target cells wasinvestigated. In the experiment, NCI-N87 tumor target cells werecultured on the dish overnight. The following day, NF-κB-uc2P/4-1BBJurkat reporter cells were added to the coated target cells in thepresence of various concentrations of the bispecific fusion polypeptidesas follows: (A) SEQ ID NOs: 9 and 10 (solid lines) or the controlantibody of SEQ ID NOs: 3 and 4 (broken line). (B) Anti-CD137 antibodySEQ ID NOs: 32 and 33. (C) Anti-CD137 antibody SEQ ID NOs: 34 and 35.The luminescence signal (RLU) represents a relative measurement of CD137pathway activation. Four parameter logistic curve analysis was performedwith GraphPad Prism software.

FIG. 18: (A) shows median tumor volume after treatment with CD137/HER2bispecifics or controls in humanized mouse tumor model. NOG mice wereengrafted with s.c. SK-OV-3 tumors which were allowed to grow to anaverage of 120 mm³. Mice were randomized into treatment groups andreceived 7×10⁶ fresh human PBMC i.v. and the molecules and dosesindicated 1 hour after PBMC injection on day 0, and again on day 7 andday 14. Each group contained 10 mice. Tumor growth was recorded twiceweekly for 20 days. (B) Shows the results of immunohistochemistry forthe human lymphocyte marker CD45 as a marker for infiltration of human Tcells on tumors from two mice that were harvested on day 20 posttreatment.

FIG. 19: (A) shows CD45, CD3 and CD8 phenotype of PMBCs of the treatmentand control groups of Example 16 taken on day 19 of that study. FIG. 19Aon the left shows the percentage of total PMBCs expressing human CD45while the FIG. 19A on the right shows the percentage of CD45-expressingPMBCs that also express CD3 and CD8. The figure shows increased CD8⁺human effector T cell expansion in the anti-CD137 mAb treatment group.(B) shows the mortality of treatment and control groups of Experiment16. Plotted values of FIG. 19B correspond to number of mice per group often that died spontaneously or needed to be sacrificed based on definedgeneral condition criteria.

FIG. 20: Mortality of treatment and control groups of Example 18.Plotted values correspond to number of mice per group of ten (SEQ IDNos: 9 and 10, 4 μg: group of nine) that died spontaneously or needed tobe sacrificed based on defined general condition criteria.

FIG. 21: Relative median tumor volume after treatment with CD137/HER2bispecifics or controls in humanized mouse tumor model. NSG mice wereengrafted with s.c. SK-OV-3 tumors which were allowed to grow to anaverage of 110 mm³. Mice were randomized into treatment groups andreceived 7×10⁶ fresh human PBMC intravenously and intraperitonealinjections of the molecules and doses indicated 1 hour after PBMCinjection on day 0, and again on day 7 and day 14. Each group contained10 mice, except for group 7 (SEQ ID Nos: 9 and 10, 4 μg) which containedonly 9 mice. Tumor growth was recorded every 3-4 days.

FIG. 22: Immunohistochemistry of human CD45-positive lymphocytes. Tumorswere excised from tumor-bearing mice and up to six tumors for each groupwere formalin-fixed, embedded in paraffin and processed forimmunohistochemistry using anti-human CD45 antibodies. CD45-positivecells were identified by 3,3′-diaminobenzidine (DAB) staining. To allowclear visualization of DAB-positivity in a greyscale image, contrast andbrightness of the images was digitally adjusted.

FIG. 23: Digital quantitation of DAB positivity of the images shown inFIG. 22. The Figure illustrates increased frequencies of hCD45-positivehuman lymphocytes in the groups treated with SEQ ID Nos: 9 and 10compared to various controls (see Example 17 for details).

FIG. 24: Phenotype of PMBCs of the treatment and control groups ofExample 18 taken on day (19) of that study. (A) Percentage of totalPMBCs expressing human CD45. (B) Percentage of CD45-expressing PMBCsthat express CD8. The Figure shows increased CD8⁺ human effector T cellexpansion in the anti-CD137 mAb (SEQ ID Nos: 32 and 33) treatment groupcompared to negative controls and SEQ ID Nos: 9 and 10 treatment groups.

IV. DETAILED DESCRIPTION OF THE DISCLOSURE

In some embodiments, the fusion polypeptide contains at least twosubunits in any order: a first subunit that comprises a full-lengthimmunoglobulin or an antigen-binding domain thereof specific forHER2/neu, and a second subunit that comprises a lipocalin muteinspecific for CD137.

In some embodiments, the fusion polypeptide also may contain a thirdsubunit. For instance, the polypeptide may contain a subunit specificfor CD137. In some embodiments, said third subunit comprises a lipocalinmutein specific for CD137.

In some embodiments, one subunit can be linked to another subunit asessentially described in FIG. 1. For example, one lipocalin mutein canbe linked, via a peptide bond, to the C-terminus of the immunoglobulinheavy chain domain (VH), the N-terminus of the VH, the C-terminus of theimmunoglobulin light chain (VL), and/or the N-terminus of the VL (cf.FIG. 1). In some particular embodiments, a lipocalin mutein subunit canbe fused at its N-terminus and/or its C-terminus to an immunoglobulinsubunit. For example, the lipocalin mutein may be linked via a peptidebond between the C-terminus of a heavy chain constant region (CH) or theC-terminus of a light chain constant region (CL) of the immunoglobulin.In some still further embodiments, the peptide bond may be anunstructured (G4S)3 linker, for example, as shown in SEQ ID NO: 19.

In this regard, one subunit may be fused at its N-terminus and/or itsC-terminus to another subunit. For example, when one subunit comprises afull-length immunoglobulin, another subunit may be linked via a peptidebond between the N-terminus of the second subunit and the C-terminus ofa heavy chain constant region (CH) of said immunoglobulin. In somefurther embodiments, the third subunit may be linked via a peptide bondbetween the N-terminus of the third binding domain and the C-terminus ofa light chain constant region (CL) of said immunoglobulin. In some stillfurther embodiments, the peptide bond may be a unstructured (G4S)3linker, for example, as shown in SEQ ID NO: 19.

In some embodiments with respect to a fusion polypeptide of thedisclosure, one of whose subunits comprises a full-lengthimmunoglobulin, while the polypeptide is simultaneously engagingHER2/neu and CD137, the Fc function of the Fc region of the full-lengthimmunoglobulin to Fc receptor-positive cell may be preserved at the sametime.

In some embodiments, the CD137-specific subunit included in a fusionpolypeptide of the disclosure may be a lipocalin mutein that is specificfor CD137, such as the lipocalin mutein of SEQ ID NO: 2. In someembodiments, the CD137-specific subunit included in a fusion polypeptideof the disclosure may be a full-length immunoglobulin or anantigen-binding domain thereof that is specific for CD137, such as amonoclonal antibody (e.g. the antibody of SEQ ID NOs: 3 and 4).

In some embodiments, the HER2/neu-specific subunit included in a fusionpolypeptide of the disclosure may be a lipocalin mutein that is specificfor HER2/neu. In some embodiments, the HER2/neu-specific subunitincluded in a fusion polypeptide of the disclosure may be a full-lengthimmunoglobulin or an antigen-binding domain thereof that is specific forHER2/neu.

In some embodiments, in a fusion polypeptide of the disclosure, aCD137-specific subunit is fused to a HER2/neu-specific subunit.

In some more specific embodiments, the HER2/neu-specific subunitcomprises a full-length immunoglobulin (such as a monoclonal antibody)or an antigen-binding domain thereof and the CD137-specific subunitcomprises a lipocalin mutein. In some embodiments, the fusionpolypeptide comprises amino acid sequences selected from the groupconsisting of SEQ ID NOs of 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13and 14 or 15 and 16.

In some other embodiments with respect to a fusion polypeptide of thedisclosure, one of whose subunits comprises a full-lengthimmunoglobulin, while the polypeptide is simultaneously engagingHER2/neu and CD137, the Fc function of the Fc region of the full-lengthimmunoglobulin to Fc receptor-positive cell may be reduced or fullysuppressed by protein engineering. This may be achieved, for example, byswitching from the IgG1 backbone to IgG4, as IgG4 is known to displayreduced Fc-gamma receptor interactions compared to IgG1. To furtherreduce the residual binding to Fc-gamma receptors, mutations may beintroduced into the IgG4 backbone such as F234A and L235A. In addition,a S228P mutation may be introduced into the IgG4 backbone to minimizethe exchange of IgG4 half-antibody. In some still further embodiments,an additional N297A mutation may be present in the immunoglobulin heavychain of the fusion polypeptide in order to remove the naturalglycosylation motif; Example 7 provides evidence that modifying thesubclass of an isotype or engineering an isotype results in loss ofFc-receptor binding.

In some other embodiments with respect to a fusion polypeptide of thedisclosure, one of whose subunits comprises a full-lengthimmunoglobulin, while the polypeptide is simultaneously engagingHER2/neu and CD137, the Fc function of the Fc region of the full-lengthimmunoglobulin to neonatal Fc receptor (FcRn)-positive cells, though theFc region may be modified, e.g., by switching the isotype or subclass ofan isotype or by engineering, e.g. by engineering an isotype asdescribed herein, is retained. Example 8 provides evidence thatFc-modified or Fc-engineered Fc regions of a fusion polypeptide of thedisclosure retain binding to FcRn.

In some embodiments, resulting from the simultaneous binding to HER2 ontumor cells and CD137 on the surface of effector cells from the immunesystem, such as T-cells or NK cells, the fusion polypeptides of thedisclosure may exhibit HER2-dependent effector-cell activation, wherebythe effector cell of the immune system actively lyses theHER2-expressing tumor cell.

In some additional embodiments, the fusion polypeptide is capable ofdemonstrating comparable or superior level of HER2-dependent CD137activation as the immunoglobulin included in such fusion polypeptide,for example, when measured in an assay demonstrating target-dependenttumor-infiltrating lymphocyte expansion ex-vivo as essentially describedin Chacon, J. A. et al., PloS one 2013 8(4):e60031. In some additionalembodiments, the fusion polypeptide is capable of demonstratingcomparable or superior level of HER2-dependent CD137 activation as theimmunoglobulin included in such fusion polypeptide, for example, whenmeasured in an in-vivo xenotransplant model of human breast cancer, asessentially described in Kohrt, H. et al, J Clin Invest. 2012 March;122(3):1066-75.

In some embodiments, the Fc portion of the immunoglobulin included in afusion polypeptide of the disclosure may contribute to maintaining theserum levels of the fusion polypeptide, critical for its stability andpersistence in the body. For example, when the Fc portion binds to Fcreceptors on endothelial cells and on phagocytes, the fusion polypeptidemay become internalized and recycled back to the blood stream, enhancingits half-life within body.

In some embodiments, a fusion polypeptide of the disclosure may be ableto bind CD137 with an EC50 value at least as good as or superior to theEC50 value of the lipocalin mutein specific for CD137 as included insuch fusion polypeptide, such as lipocalin muteins of SEQ ID NO: 2, forexample, when said lipocalin mutein and the polypeptide are measured inan ELISA assay essentially as described in Example 3.

In some embodiments, the fusion polypeptide may be able to bind CD137with an EC50 value of at least about 1 nM or even lower, such as about0.6 nM, about 0.5 nM, about 0.4 nM or about 0.3 nM, for example, whenthe polypeptide is measured in an ELISA assay essentially as describedin Example 3.

In some embodiments, a fusion polypeptide of the disclosure may be ableto bind HER2/neu with an EC50 value comparable to the EC50 value of theimmunoglobulin specific for HER2/neu as included in such fusionpolypeptide, such as the antibody having the heavy and light chainsprovided by SEQ ID NOs: 3 and 4, for example, when said immunoglobulinand the fusion polypeptide are measured in as ELISA assay essentially asdescribed in Example 2.

In another aspect, the fusion polypeptide may be able to bind HER2/neuto its ligand with an EC50 value of at least about 1 nM or even lower,such as about 0.4 nM, about 0.3 nM or about 0.2 nM, for example, whenthe polypeptide is measured in an ELISA assay essentially as describedin Example 2.

In some embodiments, the fusion polypeptides of the disclosure specificfor both CD137 and HER2/neu may be capable of simultaneously binding ofCD137 and HER2/neu, for example, when said fusion polypeptide ismeasured in an ELISA assay essentially described in Example 4.

In some embodiments, the fusion polypeptides of the disclosure may becapable of co-stimulating T-cell responses in a functional T-cellactivation assay essentially described in Example 5. In someembodiments, the fusion polypeptides of the disclosure may be able toinduce IL-2 and/or IFN gamma secretion and T cell proliferation in afunctional T-cell activation assay essentially described in Example 5.In some further embodiments, the fusion polypeptides of the disclosuremay lead to successful T-cell activation in a functional T-cellactivation assay essentially described in Example 5. In some furtherembodiments, the fusion polypeptides of the disclosure may lead to localinduction of the production of IL-2 and/or IFN gamma by T-cells in thevicinity of HER2/neu-positive tumor cells essentially as described inExample 6. “In the vicinity of HER2/neu-positive cells” when used hereinmeans a distance between a T-cell bound and a HER2/neu-positive tumorcell that are both bound, i.e. “linked” by one and the same fusionpolypeptide of the present disclosure.

A. Exemplary Immunoglobulins as Included in the Fusion Polypeptides.

In some embodiments, with respect to the fusion polypeptide, the firstbinding domain comprises a full-length immunoglobulin or anantigen-binding domain thereof specific for HER2/neu. Theimmunoglobulin, for example, may be IgG1 or IgG4. In furtherembodiments, the immunoglobulin is a monoclonal antibody againstHER2/neu. A few illustrative examples for such immunoglobulins include:Trastuzumab (trade names Herclon, Herceptin) and Pertuzumab (also called2C4, trade name Perjeta).

B. Exemplary Lipocalin Muteins as Included in the Fusion Polypeptides.

As used herein, a “lipocalin” is defined as a monomeric protein ofapproximately 18-20 kDA in weight, having a cylindrical β-pleated sheetsupersecondary structural region comprising a plurality of (preferablyeight) β-strands connected pair-wise by a plurality of (preferably four)loops at one end to define thereby a binding pocket. It is the diversityof the loops in the otherwise rigid lipocalin scaffold that gives riseto a variety of different binding modes among the lipocalin familymembers, each capable of accommodating targets of different size, shape,and chemical character (reviewed, e.g., in Flower, D. R. (1996), supra;Flower, D. R. et al. (2000), supra, or Skerra, A. (2000) Biochim.Biophys. Acta 1482, 337-350). Indeed, the lipocalin family of proteinshave naturally evolved to bind a wide spectrum of ligands, sharingunusually low levels of overall sequence conservation (often withsequence identities of less than 20%) yet retaining a highly conservedoverall folding pattern. The correspondence between positions in variouslipocalins is well known to one of skill in the art. See, for example,U.S. Pat. No. 7,250,297.

As noted above, a lipocalin is a polypeptide defined by itssupersecondary structure, namely cylindrical β-pleated sheetsupersecondary structural region comprising eight β-strands connectedpair-wise by four loops at one end to define thereby a binding pocket.The present disclosure is not limited to lipocalin muteins specificallydisclosed herein. In this regard, the disclosure relates to a lipocalinmutein having a cylindrical 1-pleated sheet supersecondary structuralregion comprising eight β-strands connected pair-wise by four loops atone end to define thereby a binding pocket, wherein at least one aminoacid of each of at least three of said four loops has been mutated andwherein said lipocalin is effective to bind CD137 with detectableaffinity.

In one particular embodiment, a lipocalin mutein disclosed herein is amutein of human tear lipocalin (TLPC or Tlc), also termed lipocalin-1,tear pre-albumin or von Ebner gland protein. The term “human tearlipocalin” or “Tlc” or “lipocalin-1” as used herein refers to the maturehuman tear lipocalin with the SWISS-PROT/UniProt Data Bank AccessionNumber P31025 (Isoform 1). The amino acid sequence shown inSWISS-PROT/UniProt Data Bank Accession Number P31025 may be used as apreferred “reference sequence”, more preferably the amino acid sequenceshown in SEQ ID NO: 18 is used as reference sequence.

In another particular embodiment, a lipocalin mutein disclosed herein isa mutein of human lipocalin 2. The term “human lipocalin 2” or “humanLcn 2” or “human NGAL” as used herein refers to the mature humanneutrophil gelatinase-associated lipocalin (NGAL) with theSWISS-PROT/UniProt Data Bank Accession Number P80188. A human lipocalin2 mutein of the disclosure may also be designated herein as “an hNGALmutein”. The amino acid sequence shown in SWISS-PROT/UniProt Data BankAccession Number P80188 may be used as a preferred “reference sequence”,more preferably the amino acid sequence shown in SEQ ID NO: 17 is usedas reference sequence.

In some embodiments, a lipocalin mutein binding CD137 with detectableaffinity may include at least one amino acid substitution of a nativecysteine residue by another amino acid, for example, a serine residue.In some other embodiments, a lipocalin mutein binding CD137 withdetectable affinity may include one or more non-native cysteine residuessubstituting one or more amino acids of a wild-type lipocalin. In afurther particular embodiment, a lipocalin mutein according to thedisclosure includes at least two amino acid substitutions of a nativeamino acid by a cysteine residue, hereby to form one or more cysteinebriges. In some embodiments, said cysteine bridge may connect at leasttwo loop regions. The definition of these regions is used herein inaccordance with Flower (Flower, 1996, supra, Flower, et al., 2000,supra) and Breustedt et al. (2005, supra). In a related embodiment, thedisclosure teaches one or more lipocalin muteins that are capable ofactivating downstream signaling pathways of CD137 by binding to CD137.

Proteins of the disclosure, which are directed against or specific forCD137, include any number of specific-binding protein muteins that arebased on a defined protein scaffold. Preferably, the number ofnucleotides or amino acids, respectively, that is exchanged, deleted orinserted is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more such as 25, 30, 35, 40, 45 or 50, with 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or 11 being preferred and 9, 10 or 11 being even morepreferred. However, it is preferred that a lipocalin mutein of thedisclosure is still capable of binding CD137.

In one aspect, the present disclosure includes various lipocalin muteinsthat bind CD137 with at least detectable affinity. In this sense, CD137can be regarded a non-natural ligand of the reference wild-typelipocalin, where “non-natural ligand” refers to a compound that does notbind to wildtype lipocalins under physiological conditions. Byengineering wildtype lipocalins with one or more mutations at certainsequence positions, the present inventors have demonstrated that highaffinity and high specificity for the non-natural ligand, CD137, ispossible. In some embodiments, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or even more nucleotide triplet(s) encoding certain sequence positionson wildtype lipocalins, a random mutagenesis may be carried out throughsubstitution at these positions by a subset of nucleotide triplets.

Further, the lipocalin muteins of the disclosure may have a mutatedamino acid residue at any one or more, including at least at any one,two, three, four, five, six, seven, eight, nine, ten, eleven or twelve,of the sequence positions corresponding to certain sequence positions ofthe linear polypeptide sequence of the reference lipocalin.

A protein of the disclosure may include the wild-type (natural) aminoacid sequence of the “parental” protein scaffold (such as a lipocalin)outside the mutated amino acid sequence positions. In some embodiments,a lipocalin mutein according to the disclosure may also carry one ormore amino acid mutations at a sequence position/positions as long assuch a mutation does, at least essentially not hamper or not interferewith the binding activity and the folding of the mutein. Such mutationscan be accomplished very easily on DNA level using established standardmethods (Sambrook, J. et al. (2001) Molecular Cloning: A LaboratoryManual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). Illustrative examples of alterations of the amino acidsequence are insertions or deletions as well as amino acidsubstitutions. Such substitutions may be conservative, i.e. an aminoacid residue is replaced with an amino acid residue of chemicallysimilar properties, in particular with regard to polarity as well assize. Examples of conservative substitutions are the replacements amongthe members of the following groups: 1) alanine, serine, and threonine;2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4)arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and6) phenylalanine, tyrosine, and tryptophan. On the other hand, it isalso possible to introduce non-conservative alterations in the aminoacid sequence. In addition, instead of replacing single amino acidresidues, it is also possible to either insert or delete one or morecontinuous amino acids of the primary structure of the human tearlipocalin as long as these deletions or insertion result in a stablefolded/functional mutein (for example, Tlc muteins with truncated N- andC-terminus). In such mutein, for instance, one or more amino acidresidues are added or deleted at the N- or C-terminus of thepolypeptide. Generally such a mutein may have about at least 70%,including at least about 80%, such as at least about 85% amino acidsequence identity, with the amino acid sequence of the mature human tearlipocalin. As an illustrative example, the present disclosure alsoencompasses Tlc muteins as defined above, in which the first fourN-terminal amino acid residues of the sequence of mature human tearlipocalin (His-His-Leu-Leu; positions 1-4) and/or the last twoC-terminal amino acid residues (Ser-Asp; positions 157-158) of thelinear polypeptide sequence of the mature human tear lipocalin have beendeleted (SEQ ID NOs: 32-38). In addition, as another illustrativeexample, the present disclosure also encompasses NGAL muteins as definedabove, in which amino acid residues (Lys-Asp-Pro, positions 46-48) ofthe linear polypeptide sequence of the mature human lipocalin 2 (hNGAL)have be deleted (SEQ ID NO: 42).

The amino acid sequence of a lipocalin mutein disclosed herein has ahigh sequence identity to the reference lipocalin when compared tosequence identities with other lipocalins. In this general context, theamino acid sequence of a lipocalin mutein of the disclosure is at leastsubstantially similar to the amino acid sequence of the referencelipocalin, with the proviso that possibly there are gaps (as definedbelow) in an alignment that are the result of additions or deletions ofamino acids. A respective sequence of a lipocalin mutein of thedisclosure, being substantially similar to the sequences of thereference lipocalin, has, in some embodiments, at least 70% identity orsequence homology, at least 75% identity or sequence homology, at least80% identity or sequence homology, at least 82% identity or sequencehomology, at least 85% identity or sequence homology, at least 87%identity or sequence homology, or at least 90% identity or sequencehomology including at least 95% identity or sequence homology, to thesequence of the reference lipocalin, with the proviso that the alteredposition or sequence is retained and that one or more gaps are possible.

As used herein, a lipocalin mutein of the disclosure “specificallybinds” a target (for example, CD137) if it is able to discriminatebetween that target and one or more reference targets, since bindingspecificity is not an absolute, but a relative property. “Specificbinding” can be determined, for example, in accordance with Westernblots, ELISA-, RIA-, ECL-, IRMA-tests, FACS, IHC and peptide scans.

In one embodiment, the lipocalin muteins of the disclosure are fused atits N-terminus and/or its C-terminus to a fusion partner which is aprotein domain that extends the serum half-life of the mutein. Infurther particular embodiments, the protein domain is a Fc part of animmunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of animmunoglobulin, an albumin binding peptide, or an albumin bindingprotein.

In another embodiment, the lipocalin muteins of the disclosure areconjugated to a compound that extends the serum half-life of the mutein.More preferably, the mutein is conjugated to a compound selected fromthe group consisting of a polyalkylene glycol molecule, ahydroethylstarch, an Fc part of an immunoglobulin, a CH3 domain of animmoglobulin, a CH4 domain of an immunoglobulin, an albumin bindingpeptide, and an albumin binding protein.

In yet another embodiment, the current disclosure relates to a nucleicacid molecule comprising a nucleotide sequence encoding a lipocalinmutein disclosed herein. The disclosure encompasses a host cellcontaining said nucleic acid molecule.

In one aspect, the present disclosure provides human lipocalin muteinsthat bind CD137 and useful applications therefor. The disclosure alsoprovides methods of making CD137 binding proteins described herein aswell as compositions comprising such proteins. CD137 binding proteins ofthe disclosure as well as compositions thereof may be used in methods ofdetecting CD137 in a sample or in methods of binding of CD137 in asubject. No such human lipocalin muteins having these features attendantto the uses provided by present disclosure have been previouslydescribed.

1. Exemplary Lipocalin Muteins Specific for CD137.

In one aspect, the present disclosure provides CD137-binding human tearlipocalin muteins.

In this regard, the disclosure provides one or more Tlc muteins that arecapable of binding CD137 with an affinity measured by a KD of about 300nM or lower and even about 100 nM or lower.

In some embodiments, such Tlc mutein comprises a mutated amino acidresidue at one or more positions corresponding to positions 5, 26-31,33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108,111, 114, 121, 133, 148, 150 and 153 of the linear polypeptide sequenceof the mature human tear lipocalin (SEQ ID NO: 18).

In some particular embodiments, such Tlc mutein may contain a mutatedamino acid residue at one or more positions corresponding to positions26-34, 55-58, 60-61, 65, 104-106 and 108 of the linear polypeptidesequence of the mature human tear lipocalin.

In further particular embodiments, such Tlc mutein may further include amutated amino acid residue at one or more positions corresponding topositions 101, 111, 114 and 153 of the linear polypeptide sequence ofthe mature human tear lipocalin.

In other particular embodiments, the Tlc may contain a mutated aminoacid residue at one or more positions corresponding to positions 5,26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106,108, 111, 114, 121, 133, 148, 150 and 153 of the linear polypeptidesequence of the mature human tear lipocalin.

In some further embodiments, the Tlc mutein may comprise at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26 or even more, mutated amino acid residues at one or moresequence positions corresponding to sequence positions 5, 26-31, 33-34,42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114,121, 133, 148, 150 and 153 of the linear polypeptide sequence of themature human tear lipocalin and wherein said polypeptide binds CD137, inparticular human CD137.

In some still further embodiments, the disclosure relates to apolypeptide, wherein said polypeptide is a Tlc mutein, in comparisonwith the linear polypeptide sequence of the mature human tear lipocalin,comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even more,mutated amino acid residues at the sequence positions 526-34, 55-58,60-61, 65, 104-106 and 108 and wherein said polypeptide binds CD137, inparticular human CD137.

In some embodiments, a lipocalin mutein according to the disclosure mayinclude at least one amino acid substitution of a native cysteineresidue by e.g. a serine residue. In some embodiments, a Tlc muteinaccording to the disclosure includes an amino acid substitution of anative cysteine residue at positions 61 and/or 153 by another amino acidsuch as a serine residue. In this context it is noted that it has beenfound that removal of the structural disulfide bond (on the level of arespective nave nucleic acid library) of wild-type tear lipocalin thatis formed by the cysteine residues 61 and 153 (cf. Breustedt, et al.,2005, supra) may provide tear lipocalin muteins that are not only stablyfolded but are also able to bind a given non-natural ligand with highaffinity. In some particular embodiments, the Tlc mutein according tothe disclosure includes the amino acid substitutions Cys 61→Ala, Phe,Lys, Arg, Thr, Asn, Gly, Gln, Asp, Asn, Leu, Tyr, Met, Ser, Pro or Trpand Cys 153→Ser or Ala. Such a substitution has proven useful to preventthe formation of the naturally occurring disulphide bridge linking Cys61 and Cys 153, and thus to facilitate handling of the mutein. However,tear lipocalin muteins that binds CD137 and that have the disulphidebridge formed between Cys 61 and Cys 153 are also part of the presentdisclosure.

In some embodiments, the elimination of the structural disulde bond mayprovide the further advantage of allowing for the (spontaneous)generation or deliberate introduction of non-natural artificialdisulfide bonds into muteins of the disclosure, thereby increasing thestability of the muteins. For example, in some embodiments, either twoor all three of the cysteine codons at position 61, 101 and 153 arereplaced by a codon of another amino acid. Further, in some embodiments,a Tlc mutein according to the disclosure includes an amino acidsubstitution of a native cysteine residue at position 101 by a serineresidue or a histidine residue.

In some embodiments, a mutein according to the disclosure includes anamino acid substitution of a native amino acid by a cysteine residue atpositions 28 or 105 with respect to the amino acid sequence of maturehuman tear lipocalin.

Further, in some embodiments, a mutein according to the disclosureincludes an amino acid substitution of a native arginine residue atpositions 111 by a proline residue. Further, in some embodiments, amutein according to the disclosure includes an amino acid substitutionof a native lysine residue at positions 114 by a tryptophan residue or aglutamic acid.

In some embodiments, a CD137-binding Tlc mutein according to thedisclosure includes, at one or more positions corresponding to positions5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101,104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of the linearpolypeptide sequence of the mature human tear lipocalin (SEQ ID NO: 18),one or more of the following mutated amino acid residues: Ala 5→Val orThr; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met31→Trp; Leu 33→Ile; Glu 34→Phe; Thr 42→Ser; Gly 46→Asp; Lys 52→Glu; Leu56→Ala; Ser 58→Asp; Arg 60 Pro; Cys 61→Ala; Lys 65→Arg or Asn; Thr71→Ala; Val 85→Asp; Lys 94→Arg or Glu; Cys 101→Ser; Glu 104→Val; Leu105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Lys121→Glu; Ala 133→Thr; Arg 148→Ser; Ser 150→Ile and Cys 153→Ser. In someembodiments, a Tlc mutein according to the disclosure includes two ormore, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, even more such as 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or all mutated amino acidresidues at these sequence positions of the mature human tear lipocalin.

In some additional embodiments, the Tlc mutein binding CD137 includesone of the following sets of amino acid substitutions in comparison withthe linear polypeptide sequence of the mature human tear lipocalin:

1. Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met31→Trp; Leu 33→Ile; Glu 34→Phe; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys61→Ala; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser;Arg 111→Pro; Lys 114→Trp; Cys 153→Ser;2. Ala 5→Thr; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Leu 56→Ala; Ser 58→Asp; Arg60→Pro; Cys 61→Ala; Lys 65→Arg; Val 85→Asp; Cys 101→Ser; Glu 104→Val;Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Lys121→Glu; Ala 133→Thr; Cys 153→Ser; 157→Pro;3. Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met31→Trp; Leu 33→Ile; Glu 34→Phe; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys61→Ala; Lys 65→Asn; Lys 94→Arg; Cys 101→Ser; Glu 104→Val; Leu 105→Cys;His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Lys 121→Glu; Ala133→Thr; Cys 153→Ser;4. Ala 5→Val; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Leu 56→Ala; Ser 58→Asp; Arg60→Pro; Cys 61→Ala; Lys 65→Arg; Lys 94→Glu; Cys 101→Ser; Glu 104→Val;Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Lys121→Glu; Ala 133→Thr; Cys 153→Ser; 157→Pro;5. Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met31→Trp; Leu 33→Ile; Glu 34→Phe; Thr 42→Ser; Leu 56→Ala; Ser 58→Asp; Arg60→Pro; Cys 61→Ala; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp;Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Ser 150→Ile; Cys 153→Ser;157→Pro;6. Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met31→Trp; Leu 33→Ile; Glu 34→Phe; Lys 52→Glu; Leu 56→Ala; Ser 58→Asp; Arg60→Pro; Cys 61→Ala; Thr 71→Ala; Cys 101→Ser; Glu 104→Val; Leu 105→Cys;His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Ala 133→Thr; Arg148→Ser; Ser 150→Ile; Cys 153→Ser; 157→Pro; or7. Ala 5→Thr; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Gly 46→Asp; Leu 56→Ala; Ser58→Asp; Arg 60→Pro; Cys 61→Ala; Thr 71→Ala; Cys 101→Ser; Glu 104→Val;Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Ser150→Ile; Cys 153→Ser; 157→Pro.

In the residual region, i.e. the region differing from sequencepositions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94,101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153, a Tlc mutein ofthe disclosure may include the wild-type (natural) amino acid sequenceoutside the mutated amino acid sequence positions.

In still further embodiments, a Tlc mutein according to the currentdisclosure has at least 70% sequence identity or at least 70% sequencehomology to the sequence of the mature human tear lipocalin (SEQ ID NO:18).

In further particular embodiments, a Tlc mutein of the disclosurecomprises an amino acid sequence as set forth in any one of SEQ ID NOs:32-38 or a fragment or variant thereof.

In further particular embodiments, a Tlc mutein of the disclosure has atleast 75%, at least 80%, at least 85% or higher sequence identity to anamino acid sequence selected from the group consisting of SEQ ID NOs:32-38.

The disclosure also includes structural homologues of a Tlc muteinhaving an amino acid sequence selected from the group consisting of SEQID NOs:32-38, which structural homologues have an amino acid sequencehomology or sequence identity of more than about 60%, preferably morethan 65%, more than 70%, more than 75%, more than 80%, more than 85%,more than 90%, more than 92% and most preferably more than 95% inrelation to said Tlc mutein.

A Tlc mutein according to the present disclosure can be obtained bymeans of mutagenesis of a naturally occurring form of human tearlipocalin. In some embodiments of the mutagenesis, a substitution (orreplacement) is a conservative substitution. Nevertheless, anysubstitution—including non-conservative substitution or one or more fromthe exemplary substitutions below—is envisaged as long as the lipocalinmutein retains its capability to bind to CD137, and/or it has a sequenceidentity to the then substituted sequence in that it is at least 60%,such as at least 65%, at least 70%, at least 75%, at least 80%, at least85% or higher sequence identity to the amino acid sequence of the maturehuman tear lipocalin (SWISS-PROT Data Bank Accession Number P31025).

In some particular embodiments, the present disclosure provides a Tlcmutein that binds CD137 with an affinity measured by a KD of about 200nM or lower.

In some additional embodiments, a Tlc mutein of the disclosure does notinterfere with the binding of CD137L to CD137.

In another aspect, the present disclosure relates to novel,specific-binding human lipocalin 2 (human Lcn2 or hNGAL) muteinsdirected against or specific for CD137.

In this regard, the disclosure provides one or more hNGAL muteins thatare capable of binding CD137 with an affinity measured by a KD of 200 nMor lower, about 140 nM or lower, about 50 nM or lower, and even about 10nM or lower. More preferably, the hNGAL muteins can have an affinitymeasured by a KD of about 5 nM or lower.

In some embodiments, an hNGAL mutein of the disclosure includes at oneor more positions corresponding to positions 28, 36, 40-41, 49, 52, 65,68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132and 134 of the linear polypeptide sequence of the mature hNGAL (SEQ IDNO: 17) a substitution.

In particular embodiments, a lipocalin mutein of the disclosurecomprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, or even more, substitution(s) at a sequenceposition corresponding to sequence position 28, 36, 40-41, 49, 52, 65,68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132and 134 of the linear polypeptide sequence of the mature hNGAL(SWISS-PROT Data Bank Accession Number P80188; SEQ ID NO: 2).Preferably, it is envisaged that the disclosure relates to a lipocalinmutein which comprises, in addition to one or more substitutions atpositions corresponding to positions 36, 87 and/or 96 of the linearpolypeptide sequence of the mature human NGAL, at one or more positionscorresponding to positions 28, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79,81, 83, 94, 100, 103, 106, 125, 127, 132 and 134 of the linearpolypeptide sequence of the mature hNGAL a substitution.

In some still further embodiments, the disclosure relates to apolypeptide, wherein said polypeptide is an hNGAL mutein, in comparisonwith the linear polypeptide sequence of the mature hNGAL (SWISS-PROTData Bank Accession Number P80188; SEQ ID NO: 17), comprising at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, or even more, mutated amino acid residues at the sequence positions28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 87, 96, 100, 103,106, 125, 127, 132 and 134, and wherein said polypeptide binds CD137, inparticular human CD137.

In some embodiments, a CD137-binding hNGAL mutein of the disclosureincludes, at any one or more of the sequence positions 28, 36, 40-41,49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106,125, 127, 132 and 134 of the linear polypeptide sequence of the maturehNGAL (SEQ ID NO: 17), one or more of the following mutated amino acidresidues: Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg or Lys; Gln49→Val, Ile, His, Ser or Asn; Tyr 52→Met; Asn 65→Asp; Ser 68→Met, Ala orGly; Leu 70→Ala, Lys, Ser or Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Met,Arg, Thr or Asn; Trp 79→Ala or Asp; Arg 81→Met, Trp or Ser; Phe 83→Leu;Cys 87→Ser; Leu 94→Phe; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu and Lys 134→Tyr.

In some embodiments, an hNGAL mutein of the disclosure includes two ormore, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, even more such as 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or all mutated amino acidresidues at these sequence positions of the mature hNGAL.

In some additional embodiments, an hNGAL mutein of the disclosure, whichbinds to CD137 includes the following amino acid replacements incomparison with the linear polypeptide sequence of the mature hNGAL:

-   -   (a) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Lys; Gln 49→Asn;        Tyr 52→Met; Ser 68→Gly; Leu 70→Thr; Arg 72→Asp; Lys 73→Asp; Asp        77→Thr; Trp 79→Ala; Arg 81→Ser; Cys 87→Ser; Asn 96→Lys; Tyr        100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr        132→Glu; Lys 134→Tyr;    -   (b) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg; Gln 49→Ile;        Tyr 52→Met; Asn 65→Asp; Ser 68→Met; Leu 70→Lys; Arg 72→Asp; Lys        73→Asp; Asp 77→Met; Trp 79→Asp; Arg 81→Trp; Cys 87→Ser; Asn        96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser        127→Phe; Tyr 132→Glu; Lys 134→Tyr;    -   (c) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg; Gln 49→Asn;        Tyr 52→Met; Asn 65→Asp; Ser 68→Ala; Leu 70→Ala; Arg 72→Asp; Lys        73→Asp; Asp 77→Thr; Trp 79→Asp; Arg 81→Trp; Cys 87→Ser; Asn        96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser        127→Phe; Tyr 132→Glu; Lys 134→Tyr;    -   (d) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Lys; Gln 49→Asn;        Tyr 52→Met; Asn 65→Asp; Ser 68→Ala; Leu 70→Ala; Arg 72→Asp; Lys        73→Asp; Asp 77→Thr; Trp 79→Asp; Arg 81→Trp; Cys 87→Ser; Asn        96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser        127→Phe; Tyr 132→Glu; Lys 134→Tyr;    -   (e) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Lys; Gln 49→Ser;        Tyr 52→Met; Asn 65→Asp; Ser 68→Gly; Leu 70→Ser; Arg 72→Asp; Lys        73→Asp; Asp 77→Thr; Trp 79→Ala; Arg 81→Met; Cys 87→Ser; Asn        96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser        127→Phe; Tyr 132→Glu; Lys 134→Tyr;    -   (f) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Lys; Gln 49→Val;        Tyr 52→Met; Asn 65→Asp; Ser 68→Gly; Leu 70→Thr; Arg 72→Asp; Lys        73→Asp; Asp 77→Arg; Trp 79→Asp; Arg 81→Ser; Cys 87→Ser; Leu        94→Phe; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys        125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr;    -   (g) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg; Gln 49→His;        Tyr 52→Met; Asn 65→Asp; Ser 68→Gly; Leu 70→Thr; Arg 72→Asp; Lys        73→Asp; Asp 77→Thr; Trp 79→Ala; Arg 81→Ser; Cys 87→Ser; Asn        96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser        127→Phe; Tyr 132→Glu; Lys 134→Tyr;    -   (h) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Lys; Gln 49→Asn;        Tyr 52→Met; Asn 65→Asp; Ser 68→Gly; Leu 70→Thr; Arg 72→Asp; Lys        73→Asp; Asp 77→Thr; Trp 79→Ala; Arg 81→Ser; Phe 83→Leu; Cys        87→Ser; Leu 94→Phe; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr        106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr; or    -   (i) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg; Gln 49→Ser;        Tyr 52→Met; Asn 65→Asp; Ser 68→Ala; Leu 70→Thr; Arg 72→Asp; Lys        73→Asp; Asp 77→Asn; Trp 79→Ala; Arg 81→Ser; Cys 87→Ser; Asn        96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser        127→Phe; Tyr 132→Glu; Lys 134→Tyr.

In the residual region, i.e. the region differing from sequencepositions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87,94, 96, 100, 103, 106, 125, 127, 132 and 134, an hNGAL mutein of thedisclosure may include the wild-type (natural) amino acid sequenceoutside the mutated amino acid sequence positions.

In another embodiment, the hNGAL mutein has at least 70% or even highersequence identity to the amino acid sequence of the mature humanlipocalin 2 (SWISS-PROT Data Bank Accession Number P80188).

In further particular embodiments, a lipocalin mutein according to thecurrent disclosure comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2 and 39-46 or a fragment or variantthereof.

The amino acid sequence of a CD137-binding hNGAL mutein of thedisclosure may have a high sequence identity, such as at least 70%, atleast 75%, at least 80%, at least 82%, at least 85%, at least 87%, atleast 90% identity, including at least 95% identity, to a sequenceselected from the group consisting of SEQ ID NOs: 2 and 39-46.

The disclosure also includes structural homologues of an hNGAL muteinhaving an amino acid sequence selected from the group consisting of SEQID NOs: 2 and 39-46, which structural homologues have an amino acidsequence homology or sequence identity of more than about 60%,preferably more than 65%, more than 70%, more than 75%, more than 80%,more than 85%, more than 90%, more than 92% and most preferably morethan 95% in relation to said hNGAL mutein.

An hNGAL mutein according to the present disclosure can be obtained bymeans of mutagenesis of a naturally occurring form of human lipocalin 2.In some embodiments of the mutagenesis, a substitution (or replacement)is a conservative substitution. Nevertheless, any substitution—includingnon-conservative substitution or one or more from the exemplarysubstitutions below—is envisaged as long as the lipocalin mutein retainsits capability to bind to CD137, and/or it has an identity to the thensubstituted sequence in that it is at least 60%, such as at least 65%,at least 70%, at least 75%, at least 80%, at least 85% or higheridentity to the amino acid sequence of the mature human lipocalin 2(SWISS-PROT Data Bank Accession Number P80188).

In some particular embodiments, the present disclosure provides an hNGALmutein that binds CD137 with an affinity measured by a KD of about 5 nMor lower.

C. Exemplary Uses, Applications and Production of the FusionPolypeptides.

In some embodiments, fusion polypeptides of the disclosure may producesynergistic effect through dual-targeting CD137 and HER2. For example,as demonstrated in ex-vivo assays and mouse models, CD137 stimulation ofNK-cells boosts the activity of Trastuzumab (Kohrt, H. et al, J ClinInvest. 2012 March; 122(3):1066-75) by enhancing NK-cell function andactivity.

Numerous possible applications for the fusion polypeptides of thedisclosure, therefore, exist in medicine.

In one aspect, the disclosure relates to the use of the fusionpolypeptides disclosed herein for detecting CD137 and HER2 in a sampleas well as a respective method of diagnosis.

In another aspect, the disclosure features the use of one or more fusionpolypeptides disclosed herein or of one or more compositions comprisingsuch polypeptides for simultaneously binding of CD137 and HER2/neu.

The present disclosure also involves the use of one or more fusionpolypeptides as described for complex formation with CD137 and HER2.

Therefore, in a still further aspect of the disclosure, the disclosedone or more fusion polypeptides are used for the detection of CD137 andHER2. Such use may include the steps of contacting one or more saidfusion polypeptides, under suitable conditions, with a sample suspectedof containing CD137 and HER2, thereby allowing formation of a complexbetween the fusion polypeptides and CD137 and HER2, and detecting thecomplex by a suitable signal. The detectable signal can be caused by alabel, as explained above, or by a change of physical properties due tothe binding, i.e. the complex formation, itself. One example is surfaceplasmon resonance, the value of which is changed during binding ofbinding partners from which one is immobilized on a surface such as agold foil.

The fusion polypeptides disclosed herein may also be used for theseparation of CD137 and HER2. Such use may include the steps ofcontacting one or more said fusion polypeptides, under suitableconditions, with a sample supposed to contain CD137 and HER2, therebyallowing formation of a complex between the fusion polypeptides andCD137 and HER2, and separating the complex from the sample.

In still another aspect, the present disclosure features a diagnostic oranalytical kit comprising a fusion polypeptide according to thedisclosure.

In addition to their use in diagnostics, in yet another aspect, thedisclosure contemplates a pharmaceutical composition comprising a fusionpolypeptide of the disclosure and a pharmaceutically acceptableexcipient.

Furthermore, the present disclosure provides fusion polypeptides thatsimultaneously bind CD137 and HER2 for use as anti-cancer agents andimmune modulators. As such the fusion polypeptides of the presentdisclosure are envisaged to be used in a method of treatment orprevention of human diseases such as a variety of tumors includingcertain aggressive types of breast cancer. Accordingly, also providedare methods of treatment or prevention of human diseases such as avariety of tumors including certain aggressive types of breast cancer ina subject in need thereof, comprising administering to said subject atherapeutically effective amount of one or more fusion polypeptides ofthe disclosure.

By simultaneously targeting tumor cells where HER2/neu is expressed andactivating natural killer (NK) cells in the host innate immune systemadjacent to such tumor cells, the fusion polypeptide of the disclosuremay increase targeted anti-tumor T cells activity, enhance anti-tumorimmunity and, at the same time, have a direct inhibiting effect on tumorgrowth, thereby produce synergistic anti-tumor results. In addition, vialocally inhibiting oncogene activity and inducing cell-mediatedcytotoxicity by NK cells, the fusion polypeptide of the disclosure mayreduce side effects of effector lymphocytes towards healthy cells, i.e.off-target toxicity.

In T cells CD137-mediated signaling leads to the recruitment of TRAFfamily members and activation of several kinases, including ASK-1, MKK,MAPK3/MAPK4, p38, and JNK/SAPK. Kinase activation is then followed bythe activation and nuclear translocation of several transcriptionfactors, including ATF-2, Jun, and NF-κB. In addition to augmentingsuboptimal T cell receptor (TCR)-induced proliferation, CD137-mediatedsignaling protects T cells, and in particular, CD8+ T cells fromactivation-induced cell death (AICD).

The present disclosure encompasses the use of a fusion polypeptide ofthe disclosure or a composition comprising such fusion polypeptide forcostimulating T-cells, and/or activating downstream signaling pathwaysof CD137 when engaging tumor cells where HER2/neu is expressed.

The present disclosure also features a method of costimulating T-cellsand/or activating downstream signaling pathways of CD137 when engagingtumor cells where HER2/neu is expressed, comprising applying one or morefusion polypeptide s of the disclosure or of one or more compositionscomprising such fusion polypeptides.

Furthermore, the present disclosure involves a method of activatingdownstream signaling pathways of CD137 when engaging tumor cells whereHER2/neu is expressed, comprising applying one or more fusionpolypeptides of the disclosure or of one or more compositions comprisingsuch fusion polypeptides.

The present disclosure also contemplates a method of inducing Tlymphocyte proliferation when engaging tumor cells where HER2/neu isexpressed, comprising applying one or more fusion polypeptides of thedisclosure or of one or more compositions comprising such fusionpolypeptides.

The present disclosure encompasses the use of a fusion polypeptide ofthe disclosure or a composition comprising such fusion polypeptide fordirecting CD137 clustering and activation on T-cells to tumor cellswhere HER2/neu is expressed.

The present disclosure further provides a method of inducing a localT-cell response in the vicinity of HER2/neu-positive tumor cells,comprising applying such fusion polypeptides. “Local” means that uponbinding T-cells via CD137 and engaging HER2/neu-positive tumor cells,T-cells produce cytokines, particularly IL-2 and/or IFN gamma invicinity of the HER2/neu-positive cell. Such cytokines reflectactivation of T-cells which may then be able to kill HER2/neu-positivetumor cells, either directly or indirectly by attracting other killercells, such as T-cells or NK cells.

In another embodiment, the present disclosure also relates to nucleicacid molecules (DNA and RNA) that include nucleotide sequences encodingthe fusion polypeptides disclosed herein. In yet another embodiment, thedisclosure encompasses a host cell containing said nucleic acidmolecule. Since the degeneracy of the genetic code permits substitutionsof certain codons by other codons specifying the same amino acid, thedisclosure is not limited to a specific nucleic acid molecule encoding afusion polypeptide as described herein but encompasses all nucleic acidmolecules that include nucleotide sequences encoding a functionalpolypeptide. In this regard, the present disclosure also relates tonucleotide sequences encoding the fusion polypeptides of the disclosure.

In some embodiments, a nucleic acid molecule encoding a lipocalin muteindisclosed in this application, such as DNA, may be “operably linked” toanother nucleic acid molecule encoding an immunoglobulin of thedisclosure to allow expression of a fusion polypeptide disclosed herein.In this regard, an operable linkage is a linkage in which the sequenceelements of one nucleic acid molecule and the sequence elements ofanother nucleic acid molecule are connected in a way that enablesexpression of the fusion polypeptide as a single polypeptide.

The disclosure also relates to a method for the production of a or afusion polypeptide of the disclosure is produced starting from thenucleic acid coding for the polypeptide or any subunit therein by meansof genetic engineering methods. In some embodiments, the method can becarried out in vivo, the polypeptide can, for example, be produced in abacterial or eucaryotic host organism and then isolated from this hostorganism or its culture. It is also possible to produce a fusionpolypeptide of the disclosure in vitro, for example by use of an invitro translation system.

When producing the fusion polypeptide in vivo, a nucleic acid encodingsuch polypeptide is introduced into a suitable bacterial or eukaryotichost organism by means of recombinant DNA technology (as alreadyoutlined above). For this purpose, the host cell is first transformedwith a cloning vector that includes a nucleic acid molecule encoding afusion polypeptide as described herein using established standardmethods. The host cell is then cultured under conditions, which allowexpression of the heterologous DNA and thus the synthesis of thecorresponding polypeptide. Subsequently, the polypeptide is recoveredeither from the cell or from the cultivation medium.

In one embodiment of the disclosure, the method includes subjecting atleast one nucleic acid molecule encoding hNGAL to mutagenesis atnucleotide triplets coding for at least one, sometimes even more, of thesequence positions corresponding to the sequence positions 28, 40-52,60, 68, 65,70,71-81, 87, 89, 96, 98, 100-106, 114, 118, 120, 125-137 and145 of the linear polypeptide sequence of hNGAL (SEQ ID NO: 17).

In addition, in some embodiments, the naturally occurring disulphidebond between Cys 76 and Cys 175 may be removed in hNGAL muteins of thedisclosure. Accordingly, such muteins can be produced in a cellcompartment having a reducing redox milieu, for example, in thecytoplasma of Gram-negative bacteria.

The disclosure also includes nucleic acid molecules encoding thelipocalin muteins of the disclosure, which include additional mutationsoutside the indicated sequence positions of experimental mutagenesis.Such mutations are often tolerated or can even prove to be advantageous,for example if they contribute to an improved folding efficiency, serumstability, thermal stability or ligand binding affinity of the lipocalinmuteins.

A nucleic acid molecule disclosed in this application may be “operablylinked” to a regulatory sequence (or regulatory sequences) to allowexpression of this nucleic acid molecule.

A nucleic acid molecule, such as DNA, is referred to as “capable ofexpressing a nucleic acid molecule” or capable “to allow expression of anucleotide sequence” if it includes sequence elements which containinformation regarding to transcriptional and/or translationalregulation, and such sequences are “operably linked” to the nucleotidesequence encoding the polypeptide. An operable linkage is a linkage inwhich the regulatory sequence elements and the sequence to be expressedare connected in a way that enables gene expression. The precise natureof the regulatory regions necessary for gene expression may vary amongspecies, but in general these regions include a promoter which, inprokaryotes, contains both the promoter per se, i.e. DNA elementsdirecting the initiation of transcription, as well as DNA elementswhich, when transcribed into RNA, will signal the initiation oftranslation. Such promoter regions normally include 5′ non-codingsequences involved in initiation of transcription and translation, suchas the −35/−10 boxes and the Shine-Dalgarno element in prokaryotes orthe TATA box, CAAT sequences, and 5′-capping elements in eukaryotes.These regions can also include enhancer or repressor elements as well astranslated signal and leader sequences for targeting the nativepolypeptide to a specific compartment of a host cell.

In addition, the 3′ non-coding sequences may contain regulatory elementsinvolved in transcriptional termination, polyadenylation or the like.If, however, these termination sequences are not satisfactory functionalin a particular host cell, then they may be substituted with signalsfunctional in that cell.

Therefore, a nucleic acid molecule of the disclosure can include aregulatory sequence, such as a promoter sequence. In some embodiments anucleic acid molecule of the disclosure includes a promoter sequence anda transcriptional termination sequence. Suitable prokaryotic promotersare, for example, the tet promoter, the lacUV5 promoter or the T7promoter. Examples of promoters useful for expression in eukaryoticcells are the SV40 promoter or the CMV promoter.

The nucleic acid molecules of the disclosure can also be part of avector or any other kind of cloning vehicle, such as a plasmid, aphagemid, a phage, a baculovirus, a cosmid or an artificial chromosome.

In one embodiment, the nucleic acid molecule is included in a phasmid. Aphasmid vector denotes a vector encoding the intergenic region of atemperent phage, such as M13 or f1, or a functional part thereof fusedto the cDNA of interest. After superinfection of the bacterial hostcells with such an phagemid vector and an appropriate helper phage (e.g.M13K07, VCS-M13 or R408) intact phage particles are produced, therebyenabling physical coupling of the encoded heterologous cDNA to itscorresponding polypeptide displayed on the phage surface (see e.g.Lowman, H. B. (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 401-424, orRodi, D. J., and Makowski, L. (1999) Curr. Opin. Biotechnol. 10, 87-93).

Such cloning vehicles can include, aside from the regulatory sequencesdescribed above and a nucleic acid sequence encoding a fusionpolypeptide as described herein, replication and control sequencesderived from a species compatible with the host cell that is used forexpression as well as selection markers conferring a selectablephenotype on transformed or transfected cells. Large numbers of suitablecloning vectors are known in the art, and are commercially available.

The DNA molecule encoding a fusion polypeptide as described herein (forexample, SEQ ID NOs: 20 and 31), and in particular a cloning vectorcontaining the coding sequence of such a polypeptide can be transformedinto a host cell capable of expressing the gene. Transformation can beperformed using standard techniques. Thus, the disclosure is alsodirected to a host cell containing a nucleic acid molecule as disclosedherein.

The transformed host cells are cultured under conditions suitable forexpression of the nucleotide sequence encoding a fusion polypeptide ofthe disclosure. Suitable host cells can be prokaryotic, such asEscherichia coli (E. coli) or Bacillus subtilis, or eukaryotic, such asSaccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells,immortalized mammalian cell lines (e.g., HeLa cells or CHO cells) orprimary mammalian cells.

In some embodiments where a lipocalin mutein of the disclosure,including as comprised in in a fusion polypeptide disclosed herein,includes intramolecular disulphide bonds, it may be preferred to directthe nascent polypeptide to a cell compartment having an oxidizing redoxmilieu using an appropriate signal sequence. Such an oxidizingenvironment may be provided by the periplasm of Gram-negative bacteriasuch as E. coli, in the extracellular milieu of Gram-positive bacteriaor in the lumen of the endoplasmatic reticulum of eukaryotic cells andusually favours the formation of structural disulphide bonds.

In some embodiments, it is also possible to produce a fusion polypeptideof the disclosure in the cytosol of a host cell, preferably E. coli. Inthis case, the polypeptide can either be directly obtained in a solubleand folded state or recovered in form of inclusion bodies, followed byrenaturation in vitro. A further option is the use of specific hoststrains having an oxidizing intracellular milieu, which may thus allowthe formation of disulfide bonds in the cytosol (Venturi et al. (2002)J. Mol. Biol. 315, 1-8.).

In some embodiments, a fusion polypeptide of the disclosure as describedherein may be not necessarily generated or produced only by use ofgenetic engineering. Rather, such polypeptide can also be obtained bychemical synthesis such as Merrifield solid phase polypeptide synthesisor by in vitro transcription and translation. It is, for example,possible that promising mutations are identified using molecularmodeling and then to synthesize the wanted (designed) mutein orpolypeptide in vitro and investigate the binding activity for a targetof interest. Methods for the solid phase and/or solution phase synthesisof proteins are well known in the art (see e.g. Bruckdorfer, T. et al.(2004) Curr. Pharm. Biotechnol. 5, 29-43).

In another embodiment, a fusion polypeptide of the disclosure may beproduced by in vitro transcription/translation employingwell-established methods known to those skilled in the art.

The skilled worker will appreciate methods useful to prepare fusionpolypeptides contemplated by the present disclosure but whose protein ornucleic acid sequences are not explicitly disclosed herein. As anoverview, such modifications of the amino acid sequence include, e.g.,directed mutagenesis of single amino acid positions in order to simplifysub-cloning of a polypeptide gene or its parts by incorporating cleavagesites for certain restriction enzymes. In addition, these mutations canalso be incorporated to further improve the affinity of a fusionpolypeptide for its targets (e.g. CD137 and HER2). Furthermore,mutations can be introduced to modulate certain characteristics of thepolypeptide such as to improve folding stability, serum stability,protein resistance or water solubility or to reduce aggregationtendency, if necessary. For example, naturally occurring cysteineresidues may be mutated to other amino acids to prevent disulphidebridge formation.

Additional objects, advantages, and features of this disclosure willbecome apparent to those skilled in the art upon examination of thefollowing Examples and the attached Figures thereof, which are notintended to be limiting. Thus, it should be understood that although thepresent disclosure is specifically disclosed by exemplary embodimentsand optional features, modification and variation of the disclosuresembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this disclosure.

V. EXAMPLES Example 1: Expression and Analysis of Fusion Polypeptides

To engage HER2 and CD137 at the same time, we generated severalrepresentative antibody-lipocalin mutein fusion polypeptides, fusingtogether the antibody having the heavy and light chains provided by SEQID NOs: 3 and 4, and the lipocalin mutein of SEQ ID NO: 2 via anunstructured (G4S)3 linker (SEQ ID NO: 19). The different formats thatwere designed are depicted in FIG. 1. Such fusion polypeptides (SEQ IDNOs: 9 and 10. SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs:15 and 16) were generated via fusion of the lipocalin mutein of SEQ IDNO: 2 to either one of the four termini of a mutated variant of theantibody having an engineered IgG4 backbone, which contains a S228Pmutation to minimize IgG4 half-antibody exchange in-vitro and in-vivo(cf. Silva 2015) as well as F234A and L235A mutations to reduce Fc-gammareceptor interactions (Alegre 1992). Furthermore, we generated thefusion polypeptide of SEQ ID NOs: 7 and 8, which, in comparison with thefusion polypeptide of SEQ ID NOs: 9 and 10 has an additional N297Amutation in the antibody heavy chain (cf. Bolt 1993) in order to removethe natural glycosylation motif. This removal could potentially furtherreduce the interaction with Fc-gamma receptors. In addition, wegenerated the fusion polypeptide of SEQ ID NOs: 5 and 6, which is adirect fusion of lipocalin mutein of SEQ ID NO: 2 to C-terminal heavychain of the antibody of SEQ ID NOs: 3 and 4 with an IgG1 background andtherefore the fusion polypeptide retains the original Fc-gammainteraction of the IgG1 antibody.

The constructs were generated by gene synthesis and cloned into amammalian expression vector. They were then transiently expressed in CHOcells. The concentration of fusion polypeptides in the cell culturemedium was measured using a ForteBio Protein A sensor (Pall Corp.) andquantified using a human IgG1 standard. The titers of the constructswere as described in Table 1 below.

TABLE 1 Expression titers Expression titer Clone Name [mg/L] SEQ ID NOs:5 and 6 262 SEQ ID NOs: 9 and 10 156 SEQ ID NOs: 13 and 14 191 SEQ IDNOs: 7 and 8 181 SEQ ID NOs: 11 and 12 204 SEQ ID NOs: 15 and 16 161

The fusion polypeptides were purified using Protein A chromatographyfollowed by size-exclusion chromatography (SEC) in phosphate-bufferedsaline (PBS). After SEC purification the fractions containing monomericprotein were pooled and analyzed again using analytical SEC. Accordingto this analysis, the fusion polypeptides were fully monomeric withoutdetectable multimeric species or aggregates.

Example 2: Specificity of Fusion Polypeptides Towards HER2

We employed an ELISA assay to determine the affinity of the fusionproteins to recombinant HER2 (Sino Biological). The target was dissolvedin PBS (5 μg/mL) and coated overnight on microtiter plates at 4° C. Theplate was washed after each incubation step with 80 μL PBS supplementedwith 0.05% (v/v) Tween 20 (PBS-T) five times. The plates were blockedwith 2% BSA (w/v) in PBS for 1 h at room temperature and subsequentlywashed. Different concentrations of the benchmark antibody (SEQ ID NOs:3 and 4, Trastuzumab or Herceptin®, Roche Diagnostics) or the fusionpolypeptides were added to the wells and incubated for 1 h at roomtemperature, followed by a wash step. Bound agents under study weredetected after incubation with 1:5000 diluted anti-human IgG Fc-HRP(#109-035-098, Jackson Laboratory) in PBS-T. After an additional washstep, fluorogenic HRP substrate (QuantaBlu, Thermo) was added to eachwell and the fluorescence intensity was detected using a fluorescencemicroplate reader.

The result of the experiment was plotted in FIG. 2, together with thefit curves resulting from a 1:1 binding sigmoidal fit, where the EC50value and the maximum signal were free parameters, and the slope wasfixed to unity. The resulting EC50 values are provided in Table 2 below,including the errors of the sigmoidal fit of the data. The observed EC50values were in a similar range for all tested fusion polypeptides(0.22-0.31 nM), and in the same range as the EC50 value for thebenchmark antibody (SEQ ID NOs: 3 and 4), which was at 0.16 nM.

TABLE 2 ELISA data for HER2 binding EC50 HER2 Agent Name [nM] SEQ IDNOs: 7 and 8 0.24 ± 0.02 SEQ ID NOs: 13 and 14 0.23 ± 0.01 SEQ ID NOs:15 and 16 0.31 ± 0.01 SEQ ID NOs: 5 and 6 0.22 ± 0.01 SEQ ID NOs: 9 and10 0.28 ± 0.01 SEQ ID NOs: 11 and 12 0.23 ± 0.01 SEQ ID NOs: 3 and 40.16 0.01

Example 3: Specificity of Fusion Polypeptides Towards CD137

We employed an ELISA assay to determine the affinity of the fusionpolypeptides and the positive control lipocalin mutein of SEQ ID NO: 2to a recombinant CD137-Fc fusion (#838-4B-100, R&D Systems). The targetwas dissolved in PBS (5 μg/mL) and coated overnight on microtiter platesat 4° C. The plate was washed after each incubation step with 80 μLPBS-T five times. The plates were blocked with 2% BSA (w/v) in PBS for 1h at room temperature and subsequently washed. Different concentrationsof the CD137-specific lipocalin mutein in monomeric form (SEQ ID NO: 2)or the fusion polypeptides were added to the wells and incubated for 1 hat room temperature, followed by a wash step. Bound agents under studywere detected after incubation for 1 h at room temperature with 1:1000diluted anti-hNGAL antibody conjugated to HRP in PBS-T. After anadditional wash step, fluorogenic HRP substrate (QuantaBlu, Thermo) wasadded to each well and the fluorescence intensity was detected using afluorescence microplate reader.

The result of the experiment is plotted was FIG. 3, together with thefit curves resulting from a 1:1 binding sigmoidal fit, where the EC50value and the maximum signal were free parameters, and the slope wasfixed to unity. The resulting EC50 values are provided in Table 3,including the errors of the sigmoidal fit of the data. The observed EC50values for all tested fusion polypeptides were nearly identical withinthe experimental error and ranged from 0.30 nM to 0.47 nM, slightlysuperior to the value obtained for the positive control lipocalin muteinof SEQ ID NO: 2, which was 0.49 nM.

TABLE 3 ELISA data for CD137 binding EC50 CD137 Agent Name [nM] SEQ IDNOs: 7 and 8 0.30 ± 0.02 SEQ ID NOs: 13 and 14 0.33 ± 0.05 SEQ ID NOs:15 and 16 0.35 ± 0.03 SEQ ID NOs: 5 and 6 0.41 ± 0.04 SEQ ID NOs: 9 and10 0.37 ± 0.06 SEQ ID NOs: 11 and 12 0.47 ± 0.06 SEQ ID NO: 2 0.49 ±0.09

Example 4: Demonstration of Simultaneous Target Binding in anELISA-Based Setting

In order to demonstrate the simultaneous binding of the fusionpolypeptides to HER2 and CD137, a dual-binding ELISA format was used.Recombinant HER2 (Sino Biological) in PBS (5 μg/mL) was coated overnighton microtiter plates at 4° C. The plate was washed five times after eachincubation step with 80 μL PBS supplemented with 0.05% (v/v) Tween 20(PBS-T) using a Biotek ELx405 select CW washer. The plates were blockedwith 2% BSA (w/v) in PBS for 1 h at room temperature and subsequentlywashed again. Different concentrations of the fusion polypeptides wereadded to the wells and incubated for 1 h at room temperature, followedby a wash step. Subsequently, biotinylated human CD137-Fc was added at aconstant concentration of 1 μg/mL in PBS-T for 1 h. After washing,Extravidin-HRP (Sigma-Adrich, 1:5000 in PBS-T) was added to the wellsfor 1 h. After an additional wash step, fluorogenic HRP substrate(QuantaBlu, Thermo) was added to each well and the fluorescenceintensity was detected using a fluorescence microplate reader.

The respective experimental data was plotted in FIG. 4. All testedfusion polypeptides showed clear binding signals with EC50 valuesranging from 1-4 nM, demonstrating that these fusion polypeptides areable to engage HER2 and CD137 simultaneously.

Example 5: Functional T-Cell Activation Assay Using Coated FusionPolypeptides

We employed a T-cell activation assay to assess the ability of thefusion polypeptide of SEQ ID NOs: 15 and 16 to co-stimulate T-cellresponses. For this purpose, the fusion polypeptide of SEQ ID NOs: 15and 16 at different concentrations was coated onto a plastic dishtogether with an anti-human CD3 antibody (OKT3, eBioscience) andpurified T-cells were subsequently incubated on the coated surface. Asreadouts, we assessed continued proliferation of the T-cells after threedays incubation using a 4 h BrdU pulse, and measured supernatantinterleukin 2 (IL-2)) levels. In the following, we provide a detaileddescription of the experiment.

Human peripheral blood mononuclear cells (PBMC) from healthy volunteerdonors were isolated from buffy coats by centrifugation through aPolysucrose density gradient (Biocoll 1.077 g/mL from Biochrom),following Biochrom's protocols. The T lymphocytes were isolated from theresulting PBMC using a Pan T-cell purification Kit (Miltenyi BiotecGmbH) and the manufacturer's protocols. Purified T-cells wereresuspended in a buffer consisting of 90% FCS and 10% DMSO, immediatelyfrozen down using liquid nitrogen and stored in liquid nitrogen untilfurther use. For the assay, T cells were thawed for 16 h and cultivatedin culture media (RPMI 1640, Life Technologies) supplemented with 10%FCS and 1% Penicillin-Streptomycin (Life Technologies).

The following procedure was performed using triplicates for eachexperimental condition. Flat-bottom tissue culture plates were coatedovernight at 4° C. using 200 μL of a mixture of 0.5 μg/mL anti-CD3antibody and the fusion polypeptide of SEQ ID NOs: 15 and 16 at aconcentration of 3 μg/mL, 10 μg/mL and 30 μg/mL. As a negative control,the anti-CD3 antibody was captured alone, i.e. without the addition ofthe fusion polypeptide of SEQ ID NOs: 15 and 16. The following day,wells were washed twice with PBS, and 100 μL of the T-cell suspension(corresponding to 5×10⁴ T cells) in culture media was added to eachwell. Plates were covered with a gas permeable seal (4titude) andincubated at 37° C. in a humidified 5% C02 atmosphere for 3 days.Subsequently, the IL-2 concentration in the supernatant, as well as cellproliferation, were assessed.

In order to quantify T-cell proliferation, the chemiluminescent cellproliferation ELISA kit based on BrdU incorporation (Roche) was usedaccording to the manufacturer's instructions. Briefly, on day 3, 10 μLof BrdU labeling solution were added to each well and proliferation wasallowed to proceed for a further 4 h at 37° C. under a humidified 5% CO₂atmosphere. Plates were centrifuged at 300 g for 10 min and supernatantsof the triplicates were pooled and immediately stored at −20° C. forlater IL-2 quantification. Plates were subsequently dried at 60° C. for1 hour. 200 μL of “FixDenat” solution were added to each well and theplates were incubated at room temperature for 30 min. Incorporated BRDUwas labeled with a peroxidase-labelled anti-BrdU antibody by 2 hincubation at room temperature. BrdU levels were assessed by quantifyinga chemiluminescent peroxidase-catalysed reaction in a PheraStar FSreader.

Human IL-2 levels in the cell culture supernatants were quantified usingthe IL-2 DuoSet DuoSet kit from R&D Systems. The procedure is carriedout and described in the following. In the first step, a 384 well platewas coated at room temperature for 2 h with 1 μg/mL “Human IL-2 CaptureAntibody” (R&D System) diluted in PBS. Subsequently, wells were washed 5times with 80 μl PBS-T (PBS containing 0.05% Tween20) using a BiotekEL405 select CW washer (Biotek). After 1 h blocking in PBS-Tadditionally containing 1% casein (w/w), pooled supernatant and aconcentration series of an IL-2 standard diluted in culture medium wereincubated in the 384-well plate overnight at 4° C. To allow fordetection and quantitation of captured IL-2, a mixture of 100 ng/mLbiotinylated goat anti-hlL-2-Bio detection antibody (R&D System) and 1μg/mL Sulfotag-labelled streptavidin (Mesoscale Discovery) were added inPBS-T containing 0.5% casein and incubated at room temperature for 1 h.After washing, 25 μL reading buffer was added to each well and theelectrochemiluminescence (ECL) signal of every well was read using aMesoscale Discovery reader. Analysis and quantification were performedusing Mesoscale Discovery software.

The result for the assessment of T-cell proliferation is shown in FIG.5. There is a significant increase in continued proliferation after 3 dincubation in the wells that were coated with 10 μg/mL and 30 μg/mL ofthe fusion polypeptide of SEQ ID NOs: 15 and 16, compared to thenegative control which did not contain the fusion polypeptide of SEQ IDNOs: 15 and 16 and where the anti-CD3 antibody was captured alone.

The result of the IL-2 measurement shows that the fusion polypeptide ofSEQ ID NOs: 15 and 16 can lead to successful T-cell activation (data notshown).

Example 6: Functional T-Cell Activation Assay Using Tumor Cell BoundFusions Polypeptides

We employed a target-cell dependent T-cell activation assay to assessthe ability of the fusion polypeptides of SEQ ID NOs: 9 and 10, SEQ IDNOs: 11 and 12, SEQ ID NOs: 13 and 14, and SEQ ID NOs: 15 and 16—capableof binding CD137 and HER2 at the same time—to co-stimulate T-cellresponses when immobilized on a HER2-positive cell line. As a negativecontrol, we employed the monospecific, HER2-binding antibody of SEQ IDNOs: 3 and 4. As a further control, the experiment was performed in thepresence of an excess of the monospecific, HER2-binding antibody of SEQID NOs: 3 and 4 in order to displace the bispecific constructs SEQ IDNOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, and SEQ IDNOs: 15 and 16 from binding to HER2-positive cells. In the experiment,an anti-human CD3 antibody (OKT3, eBioscience) was coated on a plasticculture dish, and subsequently HER2-positive SKBR3 cells were culturedon the dish overnight. The next day, purified T-cells were incubated onthe coated surface in the presence of various concentrations of thefusion polypeptides of SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQID NOs: 13 and 14, and SEQ ID NOs: 15 and 16 or the control antibody ofSEQ ID NOs: 3 and 4. As readout, we measured supernatant interleukin 2(IL-2) and interferon-γ (IFN-γ) levels. In the following, the experimentis described in detail.

Human peripheral blood mononuclear cells (PBMC) from healthy volunteerdonors were isolated from buffy coats by centrifugation through aPolysucrose density gradient (Biocoll 1.077 g/mL from Biochrom),following Biochrom's protocols. The T lymphocytes were isolated from theresulting PBMC using a Pan T-cell purification Kit (Miltenyi BiotecGmbH) and the manufacturer's protocols. Purified T-cells wereresuspended in a buffer consisting of 90% FCS and 10% DMSO, immediatelyfrozen down using liquid nitrogen and stored in liquid nitrogen untilfurther use. For the assay, T cells were thawed for 16 h and cultivatedin culture media (RPMI 1640, Life Technologies) supplemented with 10%FCS and 1% Penicillin-Streptomycin (Life Technologies).

The following procedure was performed using triplicates for eachexperimental condition. Flat-bottom tissue culture plates werepre-coated or not for 1 h at 37° C. using 200 μL of 0.25 μg/mL anti-CD3antibody. The plates were subsequently washed twice with PBS. 5×10⁴SKBR3 tumor cells per well were plated and allowed to adhere overnightat 37° C. in a humidified 5% C02 atmosphere. The SKBR3 cells had beforebeen grown in culture under standard conditions, detached using Accutaseand resuspended in culture media.

On the next days, tumor cells were treated 2 hours at 37° C. withmitomycin C (Sigma Aldrich) at a concentration of 30 μg/ml in order toblock their proliferation. Plates were washed twice with PBS, and 100 μLof the T-cell suspension (corresponding to 5×10⁴ T cells) and each ofthe four fusion polypeptides, at eleven different concentrations rangingfrom 25 μg/mL to 0.4 ng/mL, or the negative control at concentrations of25 μg/mL, 0.1 μg/mL and 0.4 ng/mL, were added to each well. The samesetup was performed in parallel, but with the addition of a finalconcentration of 50 μg/mL of the monospecific, HER2-binding antibody SEQID NOs: 3 and 4. Plates were covered with a gas permeable seal (4titude)and incubated at 37° C. in a humidified 5% C02 atmosphere for 3 days.Subsequently, IL-2 and IFN-γ concentration in the supernatant wereassessed as described below.

Human IL-2 levels in the cell culture supernatants were quantified usingthe IL-2 DuoSet kit and the IFN-γ DuoSet kit from R&D Systems,respectively. The procedure is carried out analogously for bothcytokines and described in the following for IL-2 only. In the firststep, a 384 well plate was coated at room temperature for 2 h with 1μg/mL “Human IL-2 Capture Antibody” (R&D System) diluted in PBS.Subsequently, wells were washed 5 times with 80 μl PBS-T (PBS containing0.05% Tween20) using a Biotek EL405 select CW washer (Biotek). After 1 hblocking in PBS-T additionally containing 1% casein (w/w), pooledsupernatant and a concentration series of an IL-2 standard diluted inculture medium were incubated in the 384-well plate overnight at 4° C.To allow for detection and quantitation of captured IL-2, a mixture of100 ng/mL biotinylated goat anti-hlL-2-Bio detection antibody (R&DSystem) and 1 μg/mL Sulfotag-labelled streptavidin (Mesoscale Discovery)were added in PBS-T containing 0.5% casein and incubated at roomtemperature for 1 h. After washing, 25 μL reading buffer was added toeach well and the electrochemiluminescence (ECL) signal of every wellwas read using a Mesoscale Discovery reader. Analysis and quantificationwere performed using Mesoscale Discovery software. The data was fittedwith a 1:1 binding model with EC50 value, background level and plateaulevel as free parameters, and a slope that was fixed to unity. Theinduction factor was calculated from the fitted values as the quotientof plateau level and background level.

The result of a representative experiment for the bispecific fusionpolypeptide SEQ ID NOs: 9 and 10 is depicted in FIG. 6. The datademonstrates a clear increase of supernatant levels for both IL-2 (FIG.6A) and IFN-γ (FIG. 6C) with rising concentrations of the bispecificfusion polypeptide. At low concentrations of the bispecific fusionpolypeptide, the IL-2 and IFN-γ concentration measured in thesupernatant corresponds to the background level measured for thenegative control SEQ ID NOs: 3 and 4. In the presence of an excess ofthe HER2-binder SEQ ID NOs: 3 and 4, the concentrations of both IL-2(FIG. 6B) and IFN-γ (FIG. 6D) no longer show a SEQ ID NOs: 9 and10-concentration-dependent increase, but remain invariant at thebackground level.

While the plateau levels reached in the experiment are lower for SEQ IDNOs: 11 and 12 (FIG. 7) and SEQ ID NOs: 15 and 16 (FIG. 9), overallresults regarding concentration dependent induction of IL-2 and IFN-γand blockade by an excess of SEQ ID NOs: 3 and 4 are similar. Incontrast, there is no discernible increase in IL-2 or IFN-γconcentration with increasing concentration of the polypeptide fusionSEQ ID NOs: 13 and 14 (FIG. 8).

The EC50 values and induction factors resulting from a sigmoidal fit ofthe data are provided in Table 4, including data for an independentrepeat of the experiment using a different PBMC donor. The dataindicates that the EC50 values are comparable for SEQ ID NOs: 9 and 10,SEQ ID NOs: 11 and 12 and SEQ ID NOs: 15 and 16. However, the inductionfactor decreases in the order SEQ ID NOs: 9 and 10>SEQ ID NOs: 11 and12>SEQ ID NOs: 15 and 16.

The experiment clearly demonstrates a potent functional activity of SEQID NOs: 9 and 10, SEQ ID NOs: 11 and 12 and SEQ ID NOs: 15 and 16, whileSEQ ID NOs: 13 and 14 has no activity. In light of the nearly identicalaffinities for all constructs demonstrated in the Examples 4 and 5, thishighlights the important role of construct geometry.

TABLE 4 Potency IL-2 Potency IFN-g Efficacy IL-2 Efficacy IFN-g [EC50][EC50] (max/min) (max/min) SEQ ID NOs: 9 and 10 0.49 nM (0.33/0.65) 0.29nM (0.25/0.33) 7.1 (7.5/6.7) 2.7 (2.5/2.8) SEQ ID NOs: 11 and 12 0.65 nM(0.72/0.57) 0.55 nM (0.45/0.64) 5.5 (6.5/4.4) 2.8 (2.9/2.7) SEQ ID NOs:13 and 14 — — — — SEQ ID NOs: 15 and 16 0.53 nM (0.55/0.50) 0.38 nM(0.45/0.30) 3.9 (4.1/3.7)   2 (2.1/1.8)

Example 7: Affinity to Fc-Gamma Receptors hFcγ RI/CD64 and hFcγRIIIA/CD16a

To measure the binding affinities of polypeptide fusions with anengineered, IgG4-based backbone (SEQ ID NOs: 9 and 10, SEQ ID NOs: 11and 12, SEQ ID NOs: 13 and 14, and SEQ ID NOs: 15 and 16) to Fc-gammareceptors hFcγ RI/CD64 (R&D Systems) and hFcγ RIIIA/CD16a (R&D Systems),a Surface Plasmon Resonance (SPR) based assay was employed. SEQ ID NOs:3 and 4 served as a control of a monospecific antibody with an IgG1backbone. SEQ ID NOs: 5 and 6 served as a control of a polypeptidefusion that was IgG1-based. In the SPR affinity assay, polypeptidefusions were biotinylated and captured on a sensor chip CAP using theBiotin CAPture Kit (GE Healthcare). The sensor Chip CAP waspre-immobilized with an ssDNA oligo. Undiluted Biotin CAPture Reagent(streptavidin conjugated with the complementary ss-DNA oligo) wasapplied at a flow rate of 2 μL/min for 300 s. Subsequently, 10 μg/mL ofbiotinylated polypeptide fusion was applied for 300 s at a flow rate of5 μL/min. SEQ ID NOs: 3 and 4 and the polypeptide fusions werebiotinylated by incubation with EZ-Link® NHS-PEG4-Biotin (ThermoScientific) for two hours at room temperature. The excess of non-reactedbiotin reagent was removed by loading the reaction mixture onto a Zeba™Spin Desalting Plate (Thermo Scientific). The reference channel wasloaded with Biotin CAPture Reagent only.

To determine the affinity, three dilutions of hFcγ RI/CD64 (at 40, 8 and1.6 or at 100, 25 and 6 nM) or four to five dilutions of hFcγRIIIA/CD16a (at 200, 40, 8 and 1.6 nM or at 1000, 333, 111, 37 and 12nM) were prepared in running buffer (10 mM HEPES, 150 mM NaCl, 0.05% v/vSurfactant P20, 3 mM EDTA, pH 7.4 (GE Healthcare)) and applied to thechip surface. Applying a flow rate of 30 μL/min, the sample contact timewas 180 s and dissociation time was 1800/2700 s for hFcγ RI/CD64 or 300s hFcγ RIIlA/CD16a. All measurements were performed at 25° C.Regeneration of the Sensor Chip CAP surface was achieved with aninjection of 6 M Gua-HCl with 0.25 M NaOH followed by an extra wash withrunning buffer and a stabilization period of 120 s. Prior to the proteinmeasurements three regeneration cycles were performed for conditioningpurposes. Data were evaluated with Biacore T200 Evaluation software (V2.0). Double referencing was used. For hFcγ RI/CD64 the 1:1 bindingmodel was used to fit the raw data. For hFcγ RIIIA/CD16a the SteadyState Affinity model was used to fit the raw data.

Table 5 shows the results of the fit of the data for hFcγ RI/CD64. TheIgG1-based test articles SEQ ID NOs: 3 and 4 and SEQ ID NOs: 5 and 6were both at 0.3 nM, demonstrating that hFcγ RI/CD64 binding was notaffected by fusion of SEQ ID NOs: 3 and 4 to an Anticalin protein. Thepolypeptide fusions SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ IDNOs: 13 and 14, and SEQ ID NOs: 15 and 16 showed no detectable bindingto hFcγ RI/CD64. These data demonstrate that binding to hFcγ RI/CD64 canbe reduced below detection limit by switching the isotype from IgG1 toengineered IgG4.

TABLE 5 Clone name KD [nM] SEQ ID NOs: 3 and 4 0.3 SEQ ID NOs: 5 and 60.3 SEQ ID NOs: 9 and 10 not determinable SEQ ID NOs: 11 and 12 notdeterminable SEQ ID NOs: 13 and 14 not determinable SEQ ID NOs: 15 and16 not determinable

Table 6 shows the results of the fit of the data for hFcγ RIIIA/CD16a.The resulting binding affinities to hFcγ RIIIA/CD16a of the IgG1-basedtest articles SEQ ID NOs: 3 and 4 and SEQ ID NOs: 5 and 6 werecomparable to each other and around 350 nM whereas the polypeptidefusions SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and14, and SEQ ID NOs: 15 and 16 showed no detectable binding to hFcγRIIIA/CD16a. These data demonstrate that binding to hFcγ RIIIA/CD16a canbe reduced below detection limit by switching the isotype from IgG1 toengineered IgG4.

TABLE 6 Name KD [nM] SEQ ID NOs: 3 and 4 335 ± 64 SEQ ID NOs: 5 and 6369 ± 76 SEQ ID NOs: 9 and 10 not determinable SEQ ID NOs: 11 and 12 notdeterminable SEQ ID NOs: 13 and 14 not determinable SEQ ID NOs: 15 and16 not determinable

Example 8: Affinity to Neonatal Fc Receptor

To measure the binding affinities of polypeptide fusions with anengineered, IgG4-based backbone (SEQ ID NOs: 9 and 10, SEQ ID NOs: 11and 12, SEQ ID NOs: 13 and 14, and SEQ ID NOs: 15 and 16) to theneonatal Fc receptor (FcRn, Sino Biologicals, #CT009-H08H), a SurfacePlasmon Resonance (SPR) based assay was employed. SEQ ID NOs: 3 and 4served as a control of a monospecific antibody with an IgG1 backbone.SEQ ID NOs: 5 and 6 served as a control of a polypeptide fusion that wasIgG1-based. In the SPR affinity assay, FcRn was covalently immobilizedon a CM5 sensor chip (GE Healthcare) according to the manufacturer'sinstructions. Briefly, after activating the carboxyl groups of thedextran matrix with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)and N-hydroxysuccinimide (NHS), the primary amines of the FcRn proteinwere allowed to react with the NHS ester on the surface until a signalof ˜200 RU was reached. Finally, non-reacted NHS-esters were blocked bypassing a solution of 1M ethanolamine across the surface. The flow ratethroughout the immobilization procedure was 10 μl/min.

To determine their affinity, six dilutions (1000 nM, 333 nM, 111 nM, 37nM, 12 nM and 4 nM) of all constructs were prepared in running buffer(10 mM HEPES, 150 mM NaCl, 0.05% v/v Surfactant P20, 3 mM EDTA, pH 6.0)and applied to the chip surface. Applying a flow rate of 30 μL/min, thesample contact time was 180 s and dissociation time was 30 s. Allmeasurements were performed at 25° C. Regeneration of the Sensor ChipCAP surface was achieved with an injection of 10 mM glycine pH 3.0.Prior to the protein measurements three regeneration cycles areperformed for conditioning purposes. Data were evaluated with BiacoreT200 Evaluation software (V 2.0) with double referencing. The SteadyState Affinity model was used to fit the raw data.

The resulting binding affinities of all polypeptide fusions to FcRn werein the range of 1-2 μM which demonstrates that switching the isotypefrom IgG1 to IgG4 has no detectable impact on FcRn binding.

TABLE 7 Name KD [μM] SEQ ID Nos: 3 and 4 2.0 ± 0.2 SEQ ID Nos: 5 and 61.1 ± 0.03 SEQ ID Nos: 11 and 12 1.3 ± 0.07 SEQ ID Nos: 13 and 14 1.8 ±0.04 SEQ ID Nos: 9 and 10 1.7 ± 0.05 SEQ ID Nos: 13 and 14 1.8 ± 0.01

Example 9: Functional T-Cell Activation Assay Using Tumor Cells withHigh and Low HER2 Levels

We employed a target-cell dependent T-cell activation assay to assessthe ability of the fusion polypeptide of SEQ ID NOs: 9 and 10 toco-stimulate T-cell responses as a function of HER2 expression levels ofthe target cell. For that purpose, we employed HER2-high expressingSKBR3 and BT474 cells, as well as cells expressing HER2 at a levelsimilar to healthy, HER2-expressing cells, HepG2 and MCF7. Forcomparison, we investigated the behavior of reference anti-CD137monoclonal antibodies of SEQ ID NOs: 47 and 48 and SEQ ID NOs: 49 and50. As a negative control, we employed the monospecific, HER2-bindingantibody of SEQ ID NOs: 3 and 4. As a further negative control, theexperiment was carried out without the addition of a test article(“vehicle control”). In the experiment, an anti-human CD3 antibody(OKT3, eBioscience) was coated on plastic culture dishes, andsubsequently SKBR3, BT474, HepG2 or MCF7 cells were separately culturedon the dishes overnight. The next day, purified T cells were incubatedon the coated surface in the presence of various concentrations of thefusion polypeptide of SEQ ID NOs: 9 and 10, reference antibodies SEQ IDNOs: 47 and 48 and SEQ ID NOs: 49 and 50, the control antibody of SEQ IDNOs: 3 and 4, or in the absence of added test article. As readout, wemeasured supernatant interleukin 2 (IL-2) levels. In the following, theexperiment is described in detail.

Human peripheral blood mononuclear cells (PBMC) from healthy volunteerdonors were isolated from buffy coats by centrifugation through aPolysucrose density gradient (Biocoll 1.077 g/mL from Biochrom),following Biochrom's protocols. The T lymphocytes were isolated from theresulting PBMC using a Pan T cell purification Kit (Miltenyi BiotecGmbH) and the manufacturer's protocols. Purified T cells werere-suspended in a buffer consisting of 90% FCS and 10% DMSO, immediatelyfrozen down using liquid nitrogen and stored in liquid nitrogen untilfurther use. For the assay, T cells were thawed for 16 h and cultivatedin culture media (RPMI 1640, Life Technologies) supplemented with 10%FCS and 1% Penicillin-Streptomycin (Life Technologies).

The following procedure was performed using triplicates for eachexperimental condition. Flat-bottom tissue culture plates werepre-coated or not for 1 h at 37° C. using 200 μL of 0.25 μg/mL anti-CD3antibody. The plates were subsequently washed twice with PBS. 5×10⁴target tumor cells per well were plated and allowed to adhere overnightat 37° C. in a humidified 5% C02 atmosphere. The target cells had beforebeen grown in culture under standard conditions, detached using Accutaseand re-suspended in culture media.

On the next days, tumor cells were treated for 2 hours at 37° C. withmitomycin C (Sigma Aldrich) at a concentration of 30 μg/ml in order toblock their proliferation. Plates were washed twice with PBS, and 100 μLof the T-cell suspension (corresponding to 5×10⁴ T cells) and SEQ IDNOs: 9 and 10, reference antibodies SEQ ID NOs: 47 and 48 and SEQ IDNOs: 49 and 50 or the negative control SEQ ID NOs: 3 and 4 or vehicle,at concentrations ranging from 0.05 nM to 5 nM (with the exception ofBT474, were concentrations ranged from 0.1 pM to 50 nM), were added toeach well. Plates were covered with a gas permeable seal (4titude) andincubated at 37° C. in a humidified 5% C02 atmosphere for 3 days.Subsequently, the IL-2 concentration in the supernatant was assessed asdescribed below.

Human IL-2 levels in the cell culture supernatants were quantified usingthe IL-2 DuoSet kit from R&D Systems. In the first step, a 384 wellplate was coated at room temperature for 2 h with 1 μg/mL “Human IL-2Capture Antibody” (R&D System) diluted in PBS. Subsequently, wells werewashed 5 times with 80 μl PBS-T (PBS containing 0.05% Tween20) using aBiotek EL405 select CW washer (Biotek). After 1 h blocking in PBS-Tadditionally containing 1% casein (w/w), pooled supernatant and aconcentration series of an IL-2 standard diluted in culture medium wereincubated in the 384-well plate overnight at 4° C. To allow fordetection and quantitation of captured IL-2, a mixture of 100 ng/mLbiotinylated goat anti-hlL-2-Bio detection antibody (R&D System) and 1μg/mL Sulfotag-labelled streptavidin (Mesoscale Discovery) were added inPBS-T containing 0.5% casein and incubated at room temperature for 1 h.After washing, 25 μL reading buffer was added to each well and theelectrochemiluminescence (ECL) signal of every well was read using aMesoscale Discovery reader. Analysis and quantification were performedusing Mesoscale Discovery software.

The result of a representative experiment is depicted in FIG. 11. Inthis Figure, values are plotted relative to the background IL-2production in the absence of test article, and therefore represent thefold change compared to background. While the negative control of SEQ IDNOs: 3 and 4 (FIG. 11A) does not lead to IL-2 induction on T-cells withany of the four cell lines, rising concentrations of the bispecificfusion polypeptide SEQ ID NOs: 9 and 10 (FIG. 11A) induce T-cells toproduce IL-2 in the presence of the highly HER2-expressing SKBR3 andBT474 cells. However, no IL-2 increase due to SEQ ID NOs: 9 and 10 isapparent for HepG2 and MCF7 cells. This behavior is markedly differentfrom both the first anti-CD137 antibody SEQ ID NOs: 47 and 48, whichinduces IL-2 on T-cells in the presence of all four cell lines (FIG.11B), and the second anti-CD137 antibody SEQ ID NOs: 49 and 50, whichdoes not lead to induction of IL-2 on any of the four cell lines (FIG.11C).

The experiment demonstrates that the fusion protein defined by SEQ IDNOs: 9 and 10 activates T-cells in a manner that is dependent on theHER2 density of the target cells. While the highly HER2-expressing SKBR3and BT474 cells show a clear T-cell activation as measured by IL-2production, this effect does not occur with HepG2 and MCF7 cells, whichexpress HER2 at a considerably lower level. That this effect isattributable to the HER2 density and not to a potential inhibition orlack of costimulation brought about by the cell under study whichrenders CD137 signaling ineffective becomes apparent by the fact thatthe anti-CD137 antibody SEQ ID NOs: 47 and 48 is capable of activating Tcells via CD137 signaling with all four cell types.

Example 10: 4-Week Stability Study of Fusion Polypeptides in Buffer atNeutral pH and Elevated Temperature

To investigate the stability of fusion polypeptide defined by SEQ IDNOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14 and SEQ IDNOs: 15 and 16, we employed an experiment where samples where incubatedfor 4 weeks at 40° C. in PBS, pH7.4 at a concentration of approximately20 mg/mL (range 21-23 mg/mL). For comparison, we investigated thebehavior of the polypeptide defined by SEQ ID NOs: 3 and 4 underidentical conditions. Samples were concentrated from a concentration ofaround 5 mg/mL to around 20 mg/mL using centrifugal filters(Ultracel-3K, Amicon), and a part of this concentrated sample was storedat −20° C. as reference material. 0.1 mL of the concentrated sample in0.5 mL tubes (PCR-PT, Sarstedt) were then stored in an incubator(Memmert) for 4 weeks at 40° C. To investigate the integrity andmonomeric content of the sample, it was then subjected to analyticalsize exclusion chromatography (SEC) using a Superdex200 Increase columnon an Agilent 1200 Series GPC2 System at a flow rate of 0.150 mL/min. 20μg of sample were applied to the column. Relative protein concentrationin the continuous flow-through was detected by absorption at awavelength of 280 nm.

FIG. 12 provides the results of the SEC analysis for all fusionpolypeptides and the control SEQ ID NOs: 3 and 4 as indicated. Therespective bottom and top SEC traces for each fusion polypeptidecorrespond to the reference material and the material incubated for 4weeks at 40° C., respectively. Comparing reference material andincubated material reveals that the SEC trace does not changesignificantly for either the bispecific fusion polypeptides or thecontrol SEQ ID NOs: 3 and 4, demonstrating the stability of the fusionpolypeptides against aggregation.

Example 11: Pharmacokinetics of Fusion Polypeptides in Mice

An analysis of the pharmacokinetics of fusion polypeptides defined bySEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14 andSEQ ID NOs: 15 and 16, as well as of SEQ ID NOs: 3 and 4 for reference,was performed in mice. Male CD-1 mice approximately 5 weeks of age (3mice per timepoint; Charles River Laboratories, Research Models andServices, Germany GmbH) were injected into a tail vein with a fusionpolypeptide at a dose of 10 mg/kg. The test articles were administeredas a bolus using a volume of 5 mL/kg. Plasma samples from the mice wereobtained at the timepoints of 5 min, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 4d, 8 d, 14 d and 20 d. Sufficient whole blood—taken under isofluraneanaesthesia—was collected to obtain at least 100 μL Li-Heparin plasmaper animal and time. Drug levels were detected using a Sandwich ELISAdetecting the full bispecific construct via the targets HER2 and CD137.Trastuzumab plasma levels were determined using a Sandwich ELISA withtargets HER2 and human Fc. The data were fitted using atwo-compartmental model using Prism GraphPad 5 software.

FIG. 13 shows plots of the plasma concentration over time for theconstructs SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13and 14 and SEQ ID NOs: 15 and 16, in all cases plotted together with thevalues obtained for SEQ ID NOs: 3 and 4 for reference. Thepharmacokinetics looked similar in all cases. Starting from a plasmaconcentration of around 200 μg/mL, plasma levels fell to a level ofaround 50 μg/mL within 48 hours, and then further decrease at a muchslower rate to a level of around 25 μg/mL at the end of the experimentafter 20 days. The bi-exponential decay of a two-compartmental model wassuccessfully applied to accurately describe the data, and a fit of thedata (FIG. 13, Table 8) using this model resulted in terminal half-livesof 15-21 days for the bispecific fusion polypeptides, compared to 13days for SEQ ID NOs: 3 and 4.

The data demonstrate that the bispecific fusions have long,antibody-like terminal half-lives in mice. Because the assay employed todetermine fusion polypeptide plasma concentrations requires a retainedactivity both towards HER2 and CD137, the result also demonstrates thatthe bispecific molecules remain intact and active over the time courseof 20 days.

TABLE 8 Terminal half-lives in mice obtained using a data fit based on atwo-compartmental model Construct Terminal half-life [days ± std errorof fit] SEQ ID NOs: 3 and 4 13.3 ± 1.2 SEQ ID NOs: 9 and 10 20.9 ± 3.8SEQ ID NOs: 11 and 12 19.1 ± 2.5 SEQ ID NOs: 13 and 14 14.8 ± 1.5 SEQ IDNOs: 15 and 16 15.6 ± 1.9

Example 12: Pharmacokinetics of Fusion Polypeptides in Cynomolgus Monkey

An analysis of the pharmacokinetics of fusion polypeptides defined bySEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14 andSEQ ID NOs: 15 and 16, as well as of SEQ ID NOs: 3 and 4 for reference,was performed in cynomolgus monkeys. Male cynomolgus monkeys received anintravenous infusion over 60 minutes, with a dose of 3 mg/kg testarticle. Plasma samples from the cynomolgus monkeys were obtained at thetimepoints of 15 min, 2 h, 4 h, 8 h, 24 h, 48 h, 3 d, 4 d, 5 d, 6 d, 7d, 9 d, 11 d, 14 d, 18 d, and 24 d. Drug levels were detected using aSandwich ELISA detecting the full bispecific construct via the targetsHER2 and CD137. Trastuzumab plasma levels were determined using aSandwich ELISA with targets HER2 and human Fc. The data were fittedusing a two-compartmental model using Prism GraphPad 5 software.

FIG. 14 shows semi-logarithmic plots of the plasma concentration overtime for the constructs SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQID NOs: 13 and 14 and SEQ ID NOs: 15 and 16, in all cases plottedtogether with the values obtained for SEQ ID NOs: 3 and 4 for reference.The pharmacokinetics looked similar in all cases. Starting from a plasmaconcentration of around 70 μg/mL, plasma levels fall to levels close tozero over the timecourse of 24 days. The bi-exponential decay of atwo-compartmental model was successfully applied to accurately describethe data, and a fit of the data (FIG. 14, Table 9) using this modelresulted in terminal half-lives of ranging from approximately 64 to 99hours for the bispecific fusion polypeptides, compared to eighty-sixhours for SEQ ID NOs: 3 and 4.

The data therefore demonstrate that the bispecific fusions have terminalhalf-lives in cynomolgus monkeys that are very similar to the half-lifeof the reference polypeptide SEQ ID NOs: 3 and 4.

TABLE 9 Terminal half-lives in male cynomolgus monkeys obtained using adata fit based on a two-compartmental model: Construct Terminalhalf-life [hours ± std error of fit] SEQ ID NOs: 3 and 4 86.0 ± 3.1 SEQID NOs: 9 and 10 98.7 ± 1.5 SEQ ID NOs: 11 and 12 65.2 ± 1.4 SEQ ID NOs:13 and 14 83.9 ± 3.0 SEQ ID NOs: 15 and 16 63.7 ± 0.7

Example 13: Ex Vivo T Cell Immunogenicity Assessment of FusionPolypeptides

To investigate the risk of the formation of anti-drug antibodies in man,an in vitro T cell immunogenicity assessment of the bispecific fusionpolypeptides SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13and 14 and SEQ ID NOs: 15 and 16, as well as of SEQ ID NOs: 3 and 4 forreference, the control antibody of SEQ ID NOs: 3 and 4 and the positivecontrol keyhole limpet hemocyanine (KLH) was performed. To perform theexperiment, PBMC from 32 donors selected to cover HLA allotypesreflective of the distribution in a global population were thawed,washed and seeded onto 96-well plates at a density of 3×10⁵ cells perwell. Test articles, diluted in assay media, were added to the cells ata concentration of 30 μg/mL. Assay medium alone was used as a blank, andkeyhole limpet hemocyanine (KLH) was used as a naïve positive control.PBMC were incubated for 7 days in a humidified atmosphere at 37° C. and5% CO₂. On day 7, PBMCs were labelled for surface phenotypic CD3+ andCD4+ markers and for DNA-incorporated EdU (5-ethynyl-2′deoxyuridine),used as a cell proliferation marker. The percentage of CD3⁺CD4⁺EdU⁺proliferating cells was measured using a Guava easyCyte 8HT flowcytometer and analysed using GuavaSoft InCyte software.

FIG. 15 provides the results of this assay for all 32 donors and alltest molecules under study. In FIG. 15A, the stimulation index wasplotted, which was obtained by the ratio of proliferation in thepresence vs. absence of test article. The threshold that defines aresponding donor (stimulation index >2) is indicated as a dotted line.In FIG. 15B, the number of responding donors as defined by thisthreshold was plotted. Evidently, the number of donors responding to thereference SEQ ID NOs: 3 and 4 lies at one and is therefore small, whileall 32 donors respond to the positive control KLH with strongproliferation above the threshold. For the bispecific fusionpolypeptides, the number of responding donors ranges from zero (SEQ IDNOs: 9 and 10) via one (SEQ ID NOs: 15 and 16) and two (SEQ ID NOs: 13and 14) to three (SEQ ID NOs: 11 and 12).

The experiment therefore demonstrates that the bispecific fusionpolypeptides, in particular SEQ ID NOs: 9 and 10 and SEQ ID NOs: 15 and16, induce little response in the in vitro T cell immunogenicityassessment, which indicates that the risk of inducing immunogenicresponses is low.

Example 14: Tumor Growth Inhibition by CD137/HER2 Bispecifics inHumanized Mouse Tumor Model

In order to investigate the activity of SEQ ID NOs: 9 and 10, SEQ IDNOs: 11 and 12, SEQ ID NOs: 13 and 14 and SEQ ID NOs: 32 and 33 in anin-vivo mouse model, we employed immune deficient NOG mice (Taconic,NOD/Shi-scid/IL-2Rγnull) engrafted with human SK-OV-3 tumors and humanPBMC. 4-6 week old NSG mice were subcutaneously (s.c.) injected with5×10⁶ SK-OV-3 cells in a matrigel/PBS (1:1) solution. Tumors wereallowed to grow to an average of 120 mm³ and on day 0 of the experimentmice were randomized into treatment groups according to tumor size andanimal weight. Mice were given 7×10⁶ fresh human PBMC intravenously(i.v.) into a tail vein. Mice received 20 μg or 100 μg of treatment orcontrol into the intraperitoneal cavity 1 hour after PBMC injection onday 0, and again on day 7 and day 14. The molecules under study wereIgG4 isotype control (Cat #DDXCH04P, Acris Antibodies GmbH), HER2/CD137bispecifics SEQ ID NOs: 9 and 10 (100 μg or 20 μg), SEQ ID NOs: 11 and12 (100 μg or 20 μg) and SEQ ID NOs: 13 and 14 (100 μg), or theCD137-binding benchmark antibody of SEQ ID NOs: 32 and 33 (100 μg). Eachgroup contained 10 mice with the exception of the group studying SEQ IDNOs: 32 and 33 which consisted of 7 mice. Tumor growth was recordedevery 3-4 days.

FIG. 16 shows the median tumor sizes relative to the starting volume atday 14 of the study. The best responses, ordered by strength of tumorgrowth inhibition, were achieved by SEQ ID NOs: 9 and 10 (100 μg), SEQID NOs: 9 and 10 (20 μg) and SEQ ID NOs: 11 and 12 (100 μg), while SEQID NOs: 11 and 12 at the lower dose of 20 μg, SEQ ID NOs: 13 and 14 (100μg) as well as the CD137-binding benchmark antibody of SEQ ID NOs: 32and 33 has a median response that was similar to that of the isotypecontrol.

Example 15: Investigating CD137 Pathway Activation UsingNF-κB-luc2P/4-1BB Jurkat Reporter Cells

We employed a target-cell based reporter assay to assess the ability ofthe fusion polypeptide of SEQ ID NOs: 9 and 10—capable of binding CD137and HER2 at the same time—to activate the CD137 pathway in dependence ofthe HER2 status of the target cell. For that purpose, we employed highlyHER2-expressing NCI-N87 gastric cancer cells that were mixed withNF-κB-luc2P/4-1BB Jurkat cells (Promega, CS196002) engineered tooverexpress CD137 and carrying a NF-κB Luciferase reporter gene. Forcomparison, we investigated the behavior of reference anti-CD137monoclonal antibodies of SEQ ID NOs: 32 and 33 and SEQ ID NOs: 34 and35. As a negative control, we employed the monospecific, HER2-bindingantibody of SEQ ID NOs: 3 and 4. As further control, we also assessedthe CD137 pathway activation in the absence of NCI-N87 cells for thefusion polypeptide of SEQ ID NOs: 9 and 10 and for anti-CD137 monoclonalantibodies of SEQ ID NOs: 32 and 33 and SEQ ID NOs: 34 and 35 as well asfor the monospecific, HER2-binding antibody of SEQ ID NOs: 3 and 4.Finally, the experiment was also carried out without the addition of atest article (“vehicle control”). The background signal measured in thepresence of NCI-N87 cells alone was assessed in wells where noNF-κB-luc2P/4-1BB Jurkat cells had been added. In the experiment,NCI-N87 cells were cultured on the dishes overnight. The next day,freshly thawed out NF-κB-uc2P/4-1BB Jurkat cells (Promega, CS196002)were incubated for six hours on the coated surface in the presence ofvarious concentrations of the fusion polypeptide of SEQ ID NOs: 9 and10, the reference antibodies SEQ ID NOs: 32 and 33 and SEQ ID NOs: 34and 35, the control antibody of SEQ ID NOs: 3 and 4, or in the absenceof added test article. As readout, we measured the luminescence inducedby the addition of Bio-GIo™ buffer (Promega, G7940) on the Jurkatreporter cells. In the following, the experiment is described in detail.

The following procedure was performed using triplicates for eachexperimental condition. Flat-bottom tissue culture plates were used tocoat 5×10⁴ target NCI-N87 tumor cells per well, with some wellsremaining without target cancer cells as control wells. The cells wereallowed to adhere overnight at 37° C. in a humidified 5% C02 atmosphere.The target cells had before been grown in culture under standardconditions, detached using Accutase and resuspended in culture media.

On the next day, plates were washed twice with PBS, and 50 μL of theNF-κB-luc2P/4-1BB Jurkat cells suspension (corresponding to 1.5×10⁵cells) and 25 μL of SEQ ID NOs: 9 and 10 at concentrations ranging from0.04 nM to 10 nM, reference antibodies SEQ ID NOs: 32 and 33 and SEQ IDNOs: 34 and 35 at concentrations ranging from 0.4 nM to 10 nM, thenegative control SEQ ID NOs: 3 and 4 at a concentration of 10 nM, orvehicle were added to each well. Plates were covered with a gaspermeable seal (4titude) and incubated at 37° C. in a humidified 5% C02atmosphere for 6 hours. Subsequently, 75 μL of Bio-GIo™ buffer (Promega,G7940) was added to each well containing cells (1:1 v/v) andluminescence was measured using a luminescence plate reader (Pherastar).Analysis, quantification and curve fitting were performed using GraphpadPrism software.

The result of a representative experiment is depicted in FIG. 17. Inthis Figure, plotted values are provided in relative luminescence units(RLU). Rising concentrations of the bispecific fusion polypeptide SEQ IDNOs: 9 and 10 (FIG. 17A) induce CD137 pathway activation in reporterJurkat cells in the presence of the highly HER2-expressing NCI-N87cells, in contrast to the negative control of SEQ ID NOs: 3 and 4 (FIG.16A). Furthermore, no increase luminescence is observed when the targetNCI-N87 cells are missing. This behavior is markedly different to boththe first anti-CD137 antibody SEQ ID NOs: 32 and 33, which induces CD137pathway activation in the Jurkat reporter cells both in the presence andabsence of NCI-N87 cells (FIG. 17B), and the second anti-CD137 antibodySEQ ID NOs: 34 and 35, which does not lead to CD137 pathway activationin the Jurkat reporter cells at all (FIG. 17C).

The experiment demonstrates that SEQ ID NOs: 9 and 10 activates theCD137 pathway in a manner that depends upon the presence of target cellsexpressing HER2, as no activation occurred in the absence of NCI-N87cells. These data validate that the mode of action of SEQ ID NOs: 9 and10 in T cell activation is the activation of the CD137 pathway bycrosslinking the CD137 receptor via engagement of HER2 on cancer cells.The HER2-positive cell-specific mode of action is further highlighted bycomparison to the data of the anti-CD137 antibody SEQ ID NOs: 32 and 33,which activates T cells via CD137 signaling whether target cells arepresent or not.

Example 16: Tumor Growth Inhibition by CD137/HER2 Bispecifics inHumanized Mouse Tumor Model

In a protocol similar to Example 14, immuno-compromised mice engraftedwith HER2-positive tumor cells (SKOV-3) were injected with human PBMCand treated over 3 weeks with SEQ ID NOs: 9 and 10 at a 100 μg/week or20 μg/week, anti-CD137 antibody SEQ ID NOs: 32 and 33, or controls,which were vehicle with PBMC (“PBMC only”), vehicle without PBMC (“noPBMC”) or isotype control with PBMC. Specifically, NOG mice weresubcutaneously (s.c.) injected with SK-OV-3 cells and tumors wereallowed to grow to an average of 120 mm³ prior to randomization intotreatment groups. There were 10 animals per treatment group. Mice wereengrafted with fresh human PBMC intravenously (i.v.) into a tail veinand treatment commenced 1 hour later. Mice received 3 weeklyintraperitoneal (i.p.) doses of treatment (20 μg or 100 μg) of SEQ IDNOs: 9 and 10 or SEQ ID NOs: 32 and 33 or control. Tumor growth wasrecorded twice weekly. Tumors from two mice were harvested on day 20post treatment and assessed for infiltration of human T cells byimmunohistochemistry via staining for the human lymphocyte marker CD45.

The results of the experiment are reported in FIG. 18. FIG. 18A showsmedian tumor growth over time. Data points that no longer represent thefull group size of 10 mice are connected by dotted lines. The bestresponses, ordered by strength of tumor growth inhibition, were achievedby SEQ ID NOs: 9 and 10 (100 μg), followed by the lower dose (20 μg) ofthe same antibody, while the CD137-binding benchmark antibody of SEQ IDNOs: 32 and 33 has a median response that was similar to that of theisotype control. FIG. 18B shows Immunohistochemistry of tumors afterstudy end. Sections of formalin-fixed and paraffin-embedded tumors (2per group) were stained for the human lymphocyte marker CD45; thefrequency of CD45-positive cells was quantified by dedicated software asreflected in FIG. 18B. The figure shows that SEQ ID NOs: 9 and 10 (100μg) resulted in increased frequency of human tumor infiltratinglymphocytes (TILs) while SEQ ID NOs: 9 and 10 (20 μg) and controls didnot.

The results reflect that SEQ ID NOs: 9 and 10 treatment at high dose(100 μg/wk) and low dose (20 μg/wk) resulted in stronger tumor growthinhibition (TGI) compared to isotype control or anti-CD137 benchmark.IHC staining for the human lymphocyte marker CD45 shows increasedfrequency of human TIL for high dose (100 μg) SEQ ID NOs: 9 and 10 whilelow dose (20 μg) SEQ ID NOs: 9 and 10 does not show this effect. Takentogether these data are consistent with a dual functionality of SEQ IDNOs: 9 and 10: On the one hand, the tumor-localized targeting of CD137leads to expansion of TIL's in the tumor microenvironment and suggeststumor-localized costimulatory T cell activation by SEQ ID NOs: 9 and 10while tumor growth inhibition is observed with 20 ug SEQ ID NOs: 9 and10 in the absence of TIL expansion, suggesting this activity may bedriven by HER2 antagonism.

Example 17: PBMC Phenotyping and Mortality in Humanized NOG Mouse SKOV-3Tumor Model

In order to assess the safety of SEQ ID Nos: 9 and 10, PBMCs wereisolated from mouse blood samples of the mice of Experiment 16. Thesesamples were taken on day 19 after PBMC engraftment and analysed bymulticolor FACS for human surface markers CD45, CD3 and CD8. Peripheralblood was resuspended in 10 ml 1× erythrocyte lysis buffer (0.15M NH₄Cl,10 mM KHCO₃, 0.1 mM EDTA) and lysed for 1-3 minutes at room temperaturein a 15-ml tube. Cells were centrifuged at 300×g for 10 minutes at 4°C., washed 1× with 10 ml FC-buffer (2% fetal calf serum in PBS, pH7.4)and resuspended in 200 μl of FC-buffer. Cells were transferred to a96-well plate at a density of 5×10⁵ cells/well. Cells were pelleted bycentrifugation of the plates at 400×g for 3 minutes at 4° C. and thesupernatant was removed. Fc-block antibody (10 μl/well of a 1:100dilution in buffer of 2.4G2 antibody, 0.5 mg/ml, #553142—BD Bioscience)was added to each well and plates were incubated for 15 minutes at roomtemperature. Then specific antibodies against human targets hCD45 (LifeTechnologies), hCD3 and hCD8 (both BD Bioscience) were added (0.5-1μg/sample) and plates were incubated at 4° C. for 30 minutes protectedfrom light. Following another washing step (centrifugation of the platesat 400×g for 3 min. at 4° C.), cells were resuspended in 200 μl FCBuffer for analysis with an Attune Focusing Cytometer (blue (488nm)/violet (405 nm) laser configuration). Flow cytometry data wereanalyzed with the FlowJo Data Analysis Software.

FIG. 19A shows the CD45, CD3 and CD8 phenotype of PMBCs from thetreatment and control groups of Example 16 taken on day 19 of thatstudy. FIG. 19A on the left shows the percentage of total PMBCsexpressing CD45 while the FIG. 19A on the right shows the fraction ofCD3+CD8+T effector cells in the CD45-positive cell population. Clearly,the results demonstrate that the anti-CD137 antibody SEQ ID NOs: 32 and33 treatment leads to stronger expansion of human lymphocytes in themouse peripheral blood compared to the control group or SEQ ID NOs: 9and 10, and that this expansion correlates with a strong increase in theCD8⁺ human effector T cells. FIG. 19B shows the mortality of treatmentand control groups of Experiment 16. Plotted values of FIG. 19Bcorrespond to number of mice per group of ten that died spontaneously orneeded to be sacrificed based on defined general condition criteria. Theresults reflect that Anti-CD137 mAb treatment led to acceleratedgraft-versus-host disease with significant mortality compared to controland SEQ ID NOs: 9 and 10 by the end of the study. Combined with the PBMCphenotyping data, the results indicate that the accelerated xenograftversus host disease (xGvHD) induced by anti-CD137 mAb treatment iscaused by strongly increased expansion of CD8⁺ human effector T cells inanti-CD137 group compared to the control or SEQ ID NOs: 9 and 10 groups.

Example 18: Tumor Growth Inhibition by CD137/HER2 Bispecifics inHumanized Mouse Tumor Model

In order to investigate the activity of SEQ ID NOs: 9 and 10 in anin-vivo mouse model, we employed immune deficient NOG mice (Taconic,NOD/Shi-scid/IL-2Rγnull) engrafted with human SK-OV-3 tumors and humanPBMC. The monospecific CD137-targeting antibody SEQ ID NOs: 32 and 33and the monospecific HER2-targeting construct (IgG4 backbone) SEQ IDNos: 51 and 52 were investigated in parallel to assess the effect ofmonospecific vs. bispecific targeting of the receptors HER2 and CD137.

4-6 week old NSG mice were subcutaneously (s.c.) injected with 5×10⁶SK-OV-3 cells in a matrigel/PBS (1:1) solution. Tumors were allowed togrow to an average of 110 mm³ and on day 0 of the experiment mice wererandomized into treatment groups according to tumor size and animalweight. Mice were given 7×10⁶ fresh human PBMC intravenously (i.v.) intoa tail vein. Mice received the HER2/CD137 bispecific SEQ ID NOs: 9 and10 at four different concentrations (200 μg, 100 μg, 20 μg or 4 μg),isotype control (100 μg, Cat #00004, Crown Bioscience Inc., CA),monospecific CD137-targeting antibody SEQ ID NOs: 32 and 33 (100 μg) andmonospecific HER2-targeting antibody SEQ ID Nos: 51 and 52 (80 μg) intothe intraperitoneal cavity 1 hour after PBMC injection on day 0, andagain on day 7 and day 14. Note that the dose of SEQ ID Nos: 51 and 52(80 μg) was chosen to be equimolar to that of the 100 μg SEQ ID NOs: 9and 10 group. As negative controls, groups receiving vehicle and PBMC,or vehicle only (“no PBMC”) were also included. Each group contained 10mice with the exception of the group SEQ ID NOs: 9 and 10, whichconsisted of 9 mice as one mouse succumbed on day 4 of the study due totreatment-unrelated causes. Tumor growth was recorded every 3-4 days.Statistical significance of tumor growth inhibition responses wasdetermined by a two-sided student's T-test.

FIG. 20 shows the mortality across study groups at day 20 after PBMCengraftment. Mortality caused by PBMC xenograft-vs-host-disease (xGvHD)either led to spontaneous death or to ethical sacrifice based onpredefined criteria. Strikingly, the monospecific CD137-targetingantibody SEQ ID NOs: 32 and 33 led to strongly accelerated xGvHDcompared to all other groups, with no mouse surviving to study end. Mostother groups also showed mortality before study end, with up to threemortalities at day 20. There was no apparent dose-dependency for thefour treatment groups of SEQ ID NOs: 9 and 10, and mortality was similarto the SEQ ID NOs: 51 and 52 group and the “PBMC only” group, indicatingno impact of SEQ ID NOs: 9 and 10 on onset of xGvHD.

FIG. 21 shows the absolute median tumor sizes over time. Note thatvalues are combined by dotted lines for groups that are no longercomplete due to mortality (see above). Strikingly, the isotype controlgroup led to significant tumor growth inhibition compared to the vehiclecontrol groups with and without PBMC; in the following discussion, therelevant control group for the treatment groups was therefore chosen tobe the isotype control group. Compared to this group, there was strongtumor growth inhibition for SEQ ID NOs: 9 and 10 at the weekly 200 μgand 100 μg dose as well as for SEQ ID Nos: 51 and 52 at the 80 μg dose,with all responses being similar and highly statistically significant(p<0.001). SEQ ID NOs: 9 and 10 at a dose of 20 μg weekly showed a trendtowards tumor growth inhibition compared to the isotype control group,but statistical significance was borderline (p=0.07). There was nostatistically significant difference at day 20 between the tumor growthof the isotype control group and that of either the CD137-bindingbenchmark antibody of SEQ ID NOs: 32 and 33 or of the lowest dose of theCD137/HER2 bispecific SEQ ID NOs: 9 and 10 (4 μg). Taken together, themedian tumor growth curves indicate a strong dose-dependent anti-tumoractivity of SEQ ID NOs: 9 and 10, which, taking the result for SEQ IDNOs: 51 and 52 into account, appear to be mainly driven by the anti-HER2activity of SEQ ID NOs: 9 and 10.

Example 19: Phenotyping of Tumor-Infiltrating Lymphocytes byImmunohistochemistry in Humanized Mouse Tumor Model

In a follow-up analysis of the in-vivo study described in Example 18,tumors from five or six tumor-bearing mice from each of the nine studygroups were excised on study end or ethical sacrifice and assessed forinfiltration of human T cells by immunohistochemistry via staining forthe human lymphocyte marker CD45. For that purpose, tumors wereformalin-fixed, embedded in paraffin and processed forimmunohistochemistry using anti-human CD45 antibodies. CD45-positivecells were identified by 3,3′-diaminobenzidine (DAB) staining. To allowclear visualization of DAB-positivity in a greyscale image, contrast andbrightness of the images was digitally adjusted.

An overview over all stained tumor sections is provided in FIG. 22,while FIG. 23 provides the result of a digital quantitation of thefrequency of CD45-positive cells by dedicated software. FIG. 22illustrates an evident qualitative difference between the SEQ ID NOs: 9and 10-treated groups at the weekly dosings of 200 μg, 100 μg or 20 μgcompared to all other groups: Clearly, the DAB-positivity in thecorresponding tumor slices is much stronger. The total absence ofstaining in the “no PBMC” group confirms the selectivity of the stainingprocedure. The digital quantitation (FIG. 23) confirms this qualitativefinding: With the exception of the 4 μg dosing group, the tumors fromSEQ ID NOs: 9 and 10-treated animals display a strong hCD45-positivity,indicative of a high frequency of human lymphocytes in the slides. ThehCD45-positivity is statistically significantly higher (p<0.01) than inthe control groups (“PBMC only” or isotype control) or in the groupstreated with the HER2-monospecific SEQ ID NOs: 51 and 52 or theCD137-monospecific benchmark antibody SEQ ID NOs: 32 and 33. Notably,the latter group even displays a statistically significant lower humanlymphocyte presence than that of the control groups, for examplecompared to the isotype control (p=0.02). However, this effect may bebiased by the earlier sampling that took place with the mice from thisgroup due to required ethical sacrifice.

Taken together, this data illustrates the tumor-localized costimulatorymode of action of SEQ ID NOs: 9 and 10, leading to an increasedfrequency of human lymphocytes in the tumor at weekly doses of 20 μg orhigher. Importantly, this activity is strictly driven by the bispecificactivity of SEQ ID NOs: 9 and 10, because the monospecificHER2-targeting antibody SEQ ID NOs: 51 and 52 and the monospecificCD137-targeting antibody SEQ ID NOs: 32 and 33 do not display thisactivity. On the contrary, the monospecific CD137-targeting antibody SEQID NOs: 32 and 33 even leads to a decreased frequency of humanlymphocytes compared to the negative controls.

Example 20: PBMC Phenotyping in Humanized Mouse Tumor Model

In order to further elucidate the mode of action and assess the safetyof SEQ ID Nos: 9 and 10, PBMCs were isolated from mouse blood samples ofthe mice of Experiment 18. These samples were taken from the finalbleeding after sacrifice of the mice at study end or after ethicalsacrifice and analysed by multicolor FACS for human surface markers CD45and CD8. Peripheral blood was resuspended in 10 ml 1× erythrocyte lysisbuffer (0.15M NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA) and lysed for 1-3 minutesat room temperature in a 15-ml tube. Cells were centrifuged at 300×g for10 minutes at 4° C., washed 1× with 10 ml FC-buffer (2% fetal calf serumin PBS, pH7.4) and resuspended in 200 μl of FC-buffer. Cells weretransferred to a 96-well plate at a density of 5×10⁵ cells/well. Cellswere pelleted by centrifugation of the plates at 400×g for 3 minutes at4° C. and the supernatant was removed. Fc-block antibody (10 μl/well ofa 1:100 dilution in buffer of 2.4G2 antibody, 0.5 mg/ml, #553142—BDBioscience) was added to each well and plates were incubated for 15minutes at room temperature. Then specific antibodies against humantargets hCD45 (Life Technologies) and hCD8 (BD Bioscience) were added(0.5-1 μg/sample) and plates were incubated at 4° C. for 30 minutesprotected from light. Following another washing step (centrifugation ofthe plates at 400×g for 3 minutes at 4° C.), cells were resuspended in200 μl FC Buffer for analysis with an Attune Focusing Cytometer (blue(488 nm)/violet (405 nm) laser configuration). Flow cytometry data wereanalyzed with the FlowJo Data Analysis Software.

FIG. 24 shows the CD45 and CD8 phenotype of PMBCs from the treatment andcontrol groups of Example 20 after study end. FIG. 24A shows thepercentage of total PMBCs expressing CD45 while FIG. 24B shows thefraction of CD45+CD8+T effector cells in the CD45-positive cellpopulation. Clearly, the results demonstrate that the anti-CD137antibody SEQ ID NOs: 32 and 33 treatment leads to stronger expansion ofhuman lymphocytes in the mouse peripheral blood compared to the controlgroup or all doses of SEQ ID NOs: 9 and 10, and that this expansioncorrelates with a stronger increase in the CD8⁺ human effector T cells.Combined with the mortality data shown in FIG. 20, the results indicatethat the accelerated xGvHD induced by anti-CD137 mAb treatment is causedby an increased expansion of CD8⁺ human effector T cells in theanti-CD137 group compared to the control or SEQ ID NOs: 9 and 10 groups.

Combining the evidence of Examples 18, 19 and 20, the followingconclusions can be drawn:

(i) SEQ ID NOs: 9 and 10 has a dual functionality: It leads to directtumor regression due to an anti-HER2 effect, and a tumor-localizedincrease in the density of human lymphocytes by HER2-tumor-targetlocalized CD137 targeting.

(ii) Surprisingly, the direct anti-HER2 effect is not dependent on anyFc-gamma receptor mediated effector functionality, as both theCD137/HER2 bispecific SEQ ID NOs: 9 and 10 and the HER2 monospecificantibody SEQ ID NOs: 51 and 52 should not elicit any effector functions:Both possess an IgG4 antibody backbone with additional mutations thatessentially eliminates Fc-gamma receptor interactions.

(iii) The benefit of bispecific, tumor-localized CD137 targetingregarding both efficacy and safety becomes evident by comparison withthe monospecifically CD137-targeting benchmark antibody: While theCD137/HER2 bispecific leads to a strong increase of human lymphocytes inthe tumor compared to controls, the monospecific CD137-targetingantibody even leads to a decrease compared to controls. On the otherhand, the monospecific CD137-targeting antibody leads to an expansion ofhuman CD8-positive T cells in the peripheral blood of the mice in thestudy, an effect which is not apparent for SEQ ID NOs: 9 and 10. Theperipheral expansion of CD8⁺ effector cells correlates with anaccelerated mortality of mice via xGvHD. Such toxicity brought about bysystemic activation of CD137 may also be relevant for the clinicalapplication of anti-CD137 antibodies such as SEQ ID NOs: 32 and 33.These observations strongly vouch for both an improved efficacy andsafety of SEQ ID NOs: 9 and 10 compared to monospecific anti-CD137benchmarks such as SEQ ID NOs: 32 and 33.

It is obvious to those skilled in the art that variants of the in vivomodel described in this application can be applied to show variousaspects of the in vivo efficacy of SEQ ID NOs: 9 and 10. Generally, suchmodels will be based on an engraftment with tumor cells that arepositive for the human HER2 receptor or variants thereof, which isenabled by tumor cells that are either naturally HER2-receptor positiveor made HER2-receptor positive by methods such as transfection or viraltransduction with HER2. Such cells can be either derived from immortalcancer cell lines or patient tumors. In addition, the models may rely onalloreactive or HLA-matched and tumor-reactive T cells of human ormurine origin, for example:

(I) Humanized models based on HER2-positive tumors and alloreactivePBMC. Typically, mice employed in such models will be immunocompromisedto a lesser or larger degree. Examples of a model based on an immortalcell line are provided in this application, and for example in Sanmamedet al., Cancer Res. 2015 Sep. 1; 75(17):3466-78. The latter publicationalso provides an example for a typical model based on a patient-derivedtumor cell xenograft.

(II) Humanized models based on HER2-positive tumors and monoclonal orpolyclonal T cells that recognize one or more antigens on the tumor cellline. Typically, mice employed in such models will be immunocompromisedto a lesser or larger degree. Various combinations of tumor cellspecific T cells and tumor cells are possible. Tumor cell specific Tcells may be obtained by generating partly or fully HLA-matchedmonoclonal or polyclonal tumor-reactive T cells via different protocols,for example as described in Erskine et al., J Vis Exp. 2012 Aug. 8;(66):e3683, or by transducing T cells with a natural T cell receptor,for example as described in Wang et al., Cancer Immunol Res. 2016 March;4(3):204-14, or Hirschhorn-Cymerman et al., J Exp Med. 2012 Oct. 22;209(11):2113-26. An artificial chimeric antigen receptor may also beemployed to replace the natural TCR.

(III) Patient-derived tumor cells and autologous patient-derived PBMC or(expanded) TIL. Typically, mice employed in such models will beimmunocompromised to a lesser or larger degree.

(IV) A transgenic mouse model, where the CD137 receptor is partially orfully humanized, and thus made capable of binding to SEQ ID NOs: 9 and10. Transgenic mice will be engrafted with mouse tumors that were madeto express human HER2 by cell biological methods. To increase thephysiological relevance of the model, the mouse can be additionally madetransgenic for the CD137 ligand and/or human HER2.

The models described in the above or variants thereof are expected to becapable of showing one or more of the following pharmacodynamicseffects: an increase in TIL frequency via direct or indirect enhancementof local proliferation, an increase in TIL frequency via direct orindirect suppression of lymphocyte cell death, an increase in TILactivity different from proliferation or persistence such as theproduction of proinflammatory cytokines including but not limited toIL-2, IFN-γ or TNF-α or an improved capacity to kill tumor cells asevidenced by a strong impact on tumor growth that is not due to theanti-HER2 activity of SEQ ID NOs: 9 and 10 alone. Lymphocytes affectedinclude, but are not limited to CD4- and CD8-positive T cells, NK cellsor NKT cells. Other cell types may show specific pharmacodynamicseffects, including but not limited to endothelial cells, for exampleendothelial cells of the tumor vessels. In the case of tumor endothelialcells, CD137 targeting by SEQ ID NOs: 9 and 10 may to an enhancement oftrafficking into the tumor (cf. Palazon et al., Cancer Res. 2011 Feb. 1;71(3):801-11.) via expression of targeting receptors or soluble factorsenhancing the targeting.

The models may be straightforwardly employed to study additional effectssuch as specific targeting of lymphocyte subsets, dose dependency ofpharmacodynamic and toxic effects, or treatment schedules.

Embodiments illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including”, “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present embodiments have been specificallydisclosed by preferred embodiments and optional features, modificationand variations thereof may be resorted to by those skilled in the art,and that such modifications and variations are considered to be withinthe scope of this invention. All patents, patent applications, textbooksand peer-reviewed publications described herein are hereby incorporatedby reference in their entirety. Furthermore, where a definition or useof a term in a reference, which is incorporated by reference herein, isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein applies and the definitionof that term in the reference does not apply. Each of the narrowerspecies and sub-generic groupings falling within the generic disclosurealso forms part of the invention. This includes the generic descriptionof the invention with a proviso or negative limitation removing anysubject matter from the genus, regardless of whether or not the excisedmaterial is specifically recited herein. In addition, where features aredescribed in terms of Markush groups, those skilled in the art willrecognize that the disclosure is also thereby described in terms of anyindividual member or subgroup of members of the Markush group. Furtherembodiments will become apparent from the following claims.

Equivalents: Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims. Allpublications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

1-47. (canceled)
 48. A fusion polypeptide that is capable of bindingboth CD137 and HER2/neu, wherein the fusion polypeptide comprises atleast two subunits, wherein the first subunit comprises animmunoglobulin having binding specificity for HER2/neu, and wherein thesecond subunit comprises a lipocalin mutein having binding specificityfor CD137, wherein the lipocalin mutein comprises at least 10 of thefollowing mutated amino acid residues in comparison with the linearpolypeptide sequence of mature human Lipocalin 2 (hNGAL) (SEQ ID NO:18): Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg or Lys; Gln 49→Val,Ile, His, Ser or Asn; Tyr 52→Met; Asn 65→Asp; Ser 68→Met, Ala or Gly;Leu 70→Ala, Lys, Ser or Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Met, Arg,Thr or Asn; Trp 79→Ala or Asp; Arg 81→Met, Trp or Ser; Phe 83→Leu; Cys87→Ser; Leu 94→Phe; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser;Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; and Lys 134→Tyr, and wherein thelipocalin mutein has at least 85% sequence identity to the amino acidsequence shown in SEQ ID NO:
 2. 49. The fusion polypeptide of claim 48,wherein the amino acid sequence of the lipocalin mutein comprises one ofthe following sets of mutated amino acid residues in comparison with thelinear polypeptide sequence of mature hNGAL (SEQ ID NO: 18): (a) Gln28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Lys; Gln 49→Asn; Tyr 52→Met; Ser68→Gly; Leu 70→Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Thr; Trp 79→Ala; Arg81→Ser; Cys 87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser;Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr; (b) Gln 28→His; Leu36→Gln; Ala 40→Ile; Ile 41→Arg; Gln 49→Ile; Tyr 52→Met; Asn 65→Asp; Ser68→Met; Leu 70→Lys; Arg 72→Asp; Lys 73→Asp; Asp 77→Met; Trp 79→Asp; Arg81→Trp; Cys 87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser;Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr; (c) Gln 28→His; Leu36→Gln; Ala 40→Ile; Ile 41→Arg; Gln 49→Asn; Tyr 52→Met; Asn 65→Asp; Ser68→Ala; Leu 70→Ala; Arg 72→Asp; Lys 73→Asp; Asp 77→Thr; Trp 79→Asp; Arg81→Trp; Cys 87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser;Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr; (d) Gln 28→His; Leu36→Gln; Ala 40→Ile; Ile 41→Lys; Gln 49→Asn; Tyr 52→Met; Asn 65→Asp; Ser68→Ala; Leu 70→Ala; Arg 72→Asp; Lys 73→Asp; Asp 77→Thr; Trp 79→Asp; Arg81→Trp; Cys 87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser;Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr; (e) Gln 28→His; Leu36→Gln; Ala 40→Ile; Ile 41→Lys; Gln 49→Ser; Tyr 52→Met; Asn 65→Asp; Ser68→Gly; Leu 70→Ser; Arg 72→Asp; Lys 73→Asp; Asp 77→Thr; Trp 79→Ala; Arg81→Met; Cys 87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser;Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr; (f) Gln 28→His; Leu36→Gln; Ala 40→Ile; Ile 41→Lys; Gln 49→Val; Tyr 52→Met; Asn 65→Asp; Ser68→Gly; Leu 70→Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Arg; Trp 79→Asp; Arg81→Ser; Cys 87→Ser; Leu 94→Phe; Asn 96→Lys; Tyr 100→Phe; Leu 103→His;Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr; (g) Gln28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg; Gln 49→His; Tyr 52→Met; Asn65→Asp; Ser 68→Gly; Leu 70→Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Thr; Trp79→Ala; Arg 81→Ser; Cys 87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His;Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr; (h) Gln28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Lys; Gln 49→Asn; Tyr 52→Met; Asn65→Asp; Ser 68→Gly; Leu 70→Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Thr; Trp79→Ala; Arg 81→Ser; Phe 83→Leu; Cys 87→Ser; Leu 94→Phe; Asn 96→Lys; Tyr100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr132→Glu; Lys 134→Tyr; or (i) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile41→Arg; Gln 49→Ser; Tyr 52→Met; Asn 65→Asp; Ser 68→Ala; Leu 70→Thr; Arg72→Asp; Lys 73→Asp; Asp 77→Asn; Trp 79→Ala; Arg 81→Ser; Cys 87→Ser; Asn96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe;Tyr 132→Glu; Lys 134→Tyr.
 50. The fusion polypeptide of claim 48,wherein the lipocalin mutein comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 2 and 39-46.
 51. The fusionpolypeptide of claim 48, wherein the mutein comprises an amino acidsequence having at least 95% sequence identity to the amino acidsequence shown in SEQ ID NO:
 2. 52. The fusion polypeptide of claim 48,wherein the mutein comprises the amino acid sequence of SEQ ID NO: 2.53. The fusion polypeptide of claim 48, wherein the first subunit andthe second binding domain are linked via a peptide linker between theN-terminus of the lipocalin mutein of the second subunit and theC-terminus of a heavy chain constant region (CH) of the immunoglobulinof the first subunit.
 54. The fusion polypeptide of claim 53, whereinthe peptide linker is a (G4S)3 linker.
 55. The fusion polypeptide ofclaim 48, wherein the fusion polypeptide comprises the amino acidsequence shown in SEQ ID NO:
 19. 56. The fusion polypeptide of claim 48,wherein the immunoglobulin is a monoclonal antibody.
 57. The fusionpolypeptide of claim 56, wherein the monoclonal antibody comprises theantigen-binding domain of trastuzumab or pertuzumab.
 58. The fusionpolypeptide of claim 56, wherein the monoclonal antibody has the heavyand light chains provided by SEQ ID NOs: 3 and
 4. 59. The fusionpolypeptide of claim 56, wherein the monoclonal antibody has an IgG4backbone.
 60. The fusion polypeptide of claim 59, wherein the IgG4backbone has any one of the following mutations selected from the groupconsisting of S228P, N297A, F234A and L235A.
 61. The fusion polypeptideof claim 48, wherein the fusion polypeptide has one or more of thefollowing properties: (i) it is capable of binding CD137 with an EC₅₀value comparable to or lower than the EC₅₀ value of the lipocalin muteinspecific for CD137 included in the fusion polypeptide; (ii) it iscapable of binding CD137 with an EC₅₀ value of about 1 nM or lower;(iii) it is capable of binding HER2/neu with an EC₅₀ value comparable toor lower than the EC₅₀ value of the immunoglobulin specific for HER2/neuincluded in such fusion polypeptide; (iv) it is capable of bindingHER2/neu with an EC₅₀ value of about 1 nM or lower; (v) it is capable ofsimultaneously binding CD137 and HER2/neu; (vi) it is capable ofsimultaneously binding CD137 and HER2/neu with EC₅₀ values of about 4 nMor lower.
 62. The fusion polypeptide of claim 48, wherein the fusionpolypeptide is capable of co-stimulating T cell responses.
 63. Thefusion polypeptide of claim 48, wherein the fusion polypeptide iscapable of inducing IL-2 secretion and T cell proliferation.
 64. Anucleic acid molecule comprising a nucleotide sequence encoding thefusion polypeptide of claim
 48. 65. A host cell containing a nucleicacid molecule of claim
 64. 66. A method of producing the fusionpolypeptide of claim 48, wherein the fusion polypeptide is producedstarting from a nucleic acid coding for the fusion polypeptide.
 67. Apharmaceutical composition comprising the fusion polypeptide of claim48.